Synergistic induction of insulin resistance by endothelin-1 and cAMP in 3T3-L1 adipocytes.

Both endothelin-1 (ET-1) and cAMP are implicated for inducing insulin resistance. Since we have shown previously that there is a crosstalk between ET-1 and cAMP signaling pathways in regulating glucose uptake in 3T3-L1 adipocytes, we extended our investigation in this study on whether they may have a synergistic effect on inducing insulin resistance. Our results showed that it was indeed the case. Insulin-stimulated glucose uptake, phosphorylation of PKB, IRS-1-associated PI3K, and IRS-1 tyrosine phosphorylation were all inhibited by ET-1 and 8-bromo cAMP in a synergistic manner. IRS-1 protein levels were similarly decreased by ET-1 and 8-bromo cAMP, attributable to suppressed mRNA expression. In addition, after correction for the loss in IRS-1 protein, the inhibition of insulin-stimulated IRS-1 tyrosine phosphorylation or IRS-1-associated PI3K was mainly caused by cAMP. Moreover, whereas IRS-2 protein levels were increased by cAMP or ET-1 plus cAMP, insulin-stimulated IRS-2-associated PI3K activities were abolished by both treatments. Furthermore, ET-1 and ß-adrenergic agonists had similar synergistic inhibition on insulin-stimulated glucose uptake. In conclusion, we have shown that ET-1 and cAMP may synergistically induce insulin resistance in adipocytes via inhibiting IRS-1 expression as well as insulin-stimulated IRS-1/IRS-2 activities.

The PTTG1-binding factor (PBF/PTTG1IP) regulates p53 activity in thyroid cells.

The PTTG1-binding factor (PBF/PTTG1IP) has an emerging repertoire of roles, especially in thyroid biology, and functions as a protooncogene. High PBF expression is independently associated with poor prognosis and lower disease-specific survival in human thyroid cancer. However, the precise role of PBF in thyroid tumorigenesis is unclear. Here, we present extensive evidence demonstrating that PBF is a novel regulator of p53, a tumor suppressor protein with a key role in maintaining genetic stability, which is infrequently mutated in differentiated thyroid cancer. By coimmunoprecipitation and proximity-ligation assays, we show that PBF binds specifically to p53 in thyroid cells and significantly represses transactivation of responsive promoters. Further, we identify that PBF decreases p53 stability by enhancing ubiquitination, which appears dependent on the E3 ligase activity of Mdm2. Impaired p53 function was evident in a transgenic mouse model with thyroid-specific PBF overexpression (transgenic PBF mice), which had significantly increased genetic instability as indicated by fluorescent inter simple sequence repeat-PCR analysis. Consistent with this, approximately 40% of all DNA repair genes examined were repressed in transgenic PBF primary cultures, including genes with critical roles in maintaining genomic integrity such as Mgmt, Rad51, and Xrcc3. Our data also revealed that PBF induction resulted in up-regulation of the E2 enzyme Rad6 in murine thyrocytes and was associated with Rad6 expression in human thyroid tumors. Overall, this work provides novel insights into the role of the protooncogene PBF as a negative regulator of p53 function in thyroid tumorigenesis, in which PBF is generally overexpressed and p53 mutations are rare compared with other tumor types.

Regulation of pituitary tumor transforming gene (PTTG) expression and phosphorylation in thyroid cells.

Human pituitary tumor transforming gene (hPTTG) is a multifunctional proto-oncogene implicated in the initiation and progression of several tumors. Phosphorylation of hPTTG is mediated by cyclin-dependent kinase 2 (CDC2), whereas cellular expression is regulated by specificity protein 1 (SP1). The mechanisms underlying hPTTG propagation of aberrant thyroid cell growth have not been fully defined. We set out to investigate the interplay between hPTTG and growth factors, as well as the effects of phosphorylation and SP1 regulation on hPTTG expression and function. In our study, epidermal growth factor (EGF), TGFα, and IGF-1 induced hPTTG expression and phosphorylation in thyroid cells, which was associated with activation of MAPK and phosphoinositide 3-kinase. Growth factors induced hPTTG independently of CDC2 and SP1 in thyroid carcinoma cells. Strikingly, CDC2 depletion in TPC-1 cells resulted in enhanced expression and phosphorylation of hPTTG and reduced cellular proliferation. In reciprocal experiments, hPTTG overexpression induced EGF, IGF-1, and TGFα mRNAs in primary human thyrocytes. Treatment of primary human thyrocytes with conditioned media derived from hPTTG-transfected cells resulted in autocrine upregulation of hPTTG protein, which was ameliorated by growth factor depletion or growth factor receptor tyrosine kinase inhibitors. A transgenic murine model of thyroid targeted hPTTG overexpression (hPTTG-Tg) (FVB/N strain, both sexes) demonstrated smaller thyroids with reduced cellular proliferation and enhanced secretion of Egf. In contrast, Pttg(-/-) knockout mice (c57BL6 strain, both sexes) showed reduced thyroidal Egf mRNA expression. These results define hPTTG as having a central role in thyroid autocrine signaling mechanisms via growth factors, with profound implications for promotion of transformed cell growth.

Depletion of SUMO ligase hMMS21 impairs G1 to S transition in MCF-7 breast cancer cells.

BACKGROUND: hMMS21 is a human SUMO ligase required for DNA damage repair and mitotic progression in HeLa cervical cancer cells. Owing to the diversity of cancer, we further investigated the effect of hMMS21-depletion on MCF-7 breast cancer cells. METHODS: hMMS21-depletion was achieved by RNA interference. Cellular hMMS21 and E2F1 mRNA levels were estimated by RT-PCR and real-time PCR. Cell cycle profile was assessed by flow cytometry. Western blot and co-immunoprecipitation were used to determine the protein levels of various factors involved in G1-S transition and CDK2- or CDK4-associated p21 and p27. Kinase activity of cyclin E/CDK2 was measured in anti-cyclin E immunoprecipitate. RESULTS: hMMS21-depletion induced slower cell growth and G1-S transition. While it had no effect on cyclin D1 or phospho-Rb (S807/811) levels, hMMS21-depletion provoked lower E2F1 levels and cyclin E/CDK2 activity. The decreased cyclin E/CDK2 activity correlated with increased cellular p21(CIP1) levels and CDK2-p21 association. Moreover, ectopic expression of Flag-hMMS21 but not its ligase-inactive mutant rescued the decreased growth rates of hMMS21-depletd cells. Thus, depletion of hMMS21 seems to impair G1-S transition due to lowered E2F1 protein levels and cyclin E/CDK2 activity. The decreased cyclin E/CDK2 activity is probably attributable to its greater association with p21 as a result of increased p21 levels. In addition, hMMS21-mediated sumoylation appears to be involved. GENERAL SIGNIFICANCE: This study demonstrates that hMMS21 is required for G1-S transition in breast cancer cells and implies that manipulation of hMMS21-mediated sumoylation may alter the growth rates of breast cancer cells.

Proto-oncogene PBF/PTTG1IP regulates thyroid cell growth and represses radioiodide treatment.

Pituitary tumor transforming gene (PTTG)-binding factor (PBF or PTTG1IP) is a little characterized proto-oncogene that has been implicated in the etiology of breast and thyroid tumors. In this study, we created a murine transgenic model to target PBF expression to the thyroid gland (PBF-Tg mice) and found that these mice exhibited normal thyroid function, but a striking enlargement of the thyroid gland associated with hyperplastic and macrofollicular lesions. Expression of the sodium iodide symporter (NIS), a gene essential to the radioiodine ablation of thyroid hyperplasia, neoplasia, and metastasis, was also potently inhibited in PBF-Tg mice. Critically, iodide uptake was repressed in primary thyroid cultures from PBF-Tg mice, which could be rescued by PBF depletion. PBF-Tg thyroids exhibited upregulation of Akt and the TSH receptor (TSHR), each known regulators of thyrocyte proliferation, along with upregulation of the downstream proliferative marker cyclin D1. We extended and confirmed findings from the mouse model by examining PBF expression in human multinodular goiters (MNG), a hyperproliferative thyroid disorder, where PBF and TSHR was strongly upregulated relative to normal thyroid tissue. Furthermore, we showed that depleting PBF in human primary thyrocytes was sufficient to increase radioiodine uptake. Together, our findings indicate that overexpression of PBF causes thyroid cell proliferation, macrofollicular lesions, and hyperplasia, as well as repression of the critical therapeutic route for radioiodide uptake.

A novel cross-talk between endothelin-1 and cyclic AMP signaling pathways in the regulation of GLUT1 transcription in 3T3-L1 adipocytes.

We showed previously that chronic exposure to both endothelin-1 (ET-1) and cAMP resulted in a synergistic increase in Glut1 transcription in 3T3-L1 adipocytes via a protein kinase C (PKC)-dependent mechanism. In the present study, we further examined the molecular mechanism involved. Employing transient transfections with Glut1 promoter/enhancer -luciferase reporter and several dominant negative or constitutively active PKC mutants, we identified PKCε as the responsible PKC. Investigation with deletion and mutation mutants of the promoter/enhancer reporter suggested that Sp1, CREB and AP-1 responsive elements on enhancer 2 were involved. Furthermore, chromatin immunoprecipitation and co-immunoprecipitation analysis were applied to characterize the interactions between these transcription factors and their bindings to enhancer 2 in vivo. The results indicate that there are both negative and positive interactions between ET-1 and cAMP signaling pathways. On the one hand, cAMP inhibits ET-1 induced NF-κB activation required for ET-1-stimulated Glut1 transcription; on the other hand, cAMP, via sustained CREB phosphorylation, may activate AP-1 and cooperate with ET-1-activated PKCε to enhance Sp1 expression and consequently to generate a stable enhancer 2-bound Sp1/pCREB/AP-1 complex, which can strongly facilitate Glut1 transcription more than the additive effect of ET-1 and cAMP alone.

Titanium versus autograft ossiculoplasty.

CONCLUSION: In this comparative series, hearing results were superior with titanium compared with autograft ossiculoplasty in the absence of a stapes superstructure. However, in the presence of a stapes superstructure, titanium ossiculoplasties gave superior results to autografts only when comparing an air-bone gap of < 10 dB. OBJECTIVE: To compare the hearing outcomes of autograft versus titanium ossiculoplasty at 1 year. METHODS: Two consecutive groups of patients with chronic suppurative otitis media with and without cholesteatoma suitable for ossiculoplasty, either primarily or as a staged procedure, were recruited for the study. A total of 52 consecutive patients who underwent an autograft ossiculoplasty were compared with 51 consecutive patients who underwent a titanium ossiculoplasty. Hearing results were statistically compared at 1 year between the two groups using the four frequency average (FFA) of 0.5/1/2/4 kHz and the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) four frequency average of 0.5/1/2/3 kHz. The results were analysed statistically. RESULTS: A statistically significant number of titanium TORP ossiculoplasties achieved an air-bone gap closure to within < 20 dB compared with the autograft equivalent group (p = 0.039 FFA; p = 0.016 AAO-HNS). The number of titanium PORP ossiculoplasties achieving an air-bone gap closure to within < 10 dB compared with the autograft equivalent group was also statistically significant (p = 0.006 FFA; p = 0.002 AAO-HNS).

A novel mechanism of sodium iodide symporter repression in differentiated thyroid cancer.

Differentiated thyroid cancers and their metastases frequently exhibit reduced iodide uptake, impacting on the efficacy of radioiodine ablation therapy. PTTG binding factor (PBF) is a proto-oncogene implicated in the pathogenesis of thyroid cancer. We recently reported that PBF inhibits iodide uptake, and have now elucidated a mechanism by which PBF directly modulates sodium iodide symporter (NIS) activity in vitro. In subcellular localisation studies, PBF overexpression resulted in the redistribution of NIS from the plasma membrane into intracellular vesicles, where it colocalised with the tetraspanin CD63. Cell-surface biotinylation assays confirmed a reduction in plasma membrane NIS expression following PBF transfection compared with vector-only treatment. Coimmunoprecipitation and GST-pull-down experiments demonstrated a direct interaction between NIS and PBF, the functional consequence of which was assessed using iodide-uptake studies in rat thyroid FRTL-5 cells. PBF repressed iodide uptake, whereas three deletion mutants, which did not localise within intracellular vesicles, lost the ability to inhibit NIS activity. In summary, we present an entirely novel mechanism by which the proto-oncogene PBF binds NIS and alters its subcellular localisation, thereby regulating its ability to uptake iodide. Given that PBF is overexpressed in thyroid cancer, these findings have profound implications for thyroid cancer ablation using radioiodine.

Endothelin-1 stimulates interleukin-6 secretion from 3T3-L1 adipocytes.

BACKGROUND: Since both endothelin-1 (ET-1) and interleukin-6 (IL-6) may induce insulin resistance and adipose tissue is a major contributor of circulating IL-6, we examined the effects of ET-1 on IL-6 secretion from 3T3-L1 adipocytes. METHODS: IL-6 release was measured by ELISA. RT-PCR and real-time PCR analyses were used to determine cellular IL-6 mRNA levels. A luciferase reporter driven by promoter (-1310/+198) of mouse IL-6 gene was transfected into 3T3-L1 adipocytes to monitor IL-6 transcription. RESULTS: Treatment of adipocytes with ET-1 dose- and time-dependently increased IL-6 secretion. The stimulatory effect of ET-1 on IL-6 secretion was abolished by actinomycin D and ET-1 induced an increase in IL-6 mRNA levels. ET-1 was able to enhance the IL-6 promoter activity and its stimulatory effect was inhibited by GF109203X, U0126, salicylate, dominant negative CREB and mithramycin A. Thus it appears that ET-1 may stimulate IL-6 secretion mainly through an enhanced IL-6 transcription, by a mechanism involving both protein kinase C and p42/p44 mitogen-activated protein kinase, and probably downstream NF-kappaB, CREB and Sp1 transcription factors. GENERAL SIGNIFICANCE: This study demonstrates that ET-1 is able to increase IL-6 secretion from adipocytes and raises the possibility that ET-1-induced insulin resistance may be mediated by IL-6.

Endothelin-1 induces glut1 transcription through enhanced interaction between Sp1 and NF-kappaB transcription factors.

We have shown previously that endothelin-1 (ET-1) induction of Glut1 transcription is mediated by ET-1 responsive elements on enhancer 2, via both protein kinase Cepsilon (PKCepsilon)- and p42/p44 mitogen-activated protein kinase (MAPK)-dependent pathways. In the present study, we further explored the molecular mechanism involved. By using mutation constructs of luciferase reporter driven by Glut1 promoter/enhancers, chromatin immunoprecipitation and co-immunoprecipitation experiments, we were able to demonstrate that cooperative interaction between NF-kappaB and Sp1 were required to enhance Glut1 transcription in response to ET-1. While ET-1 may induce Sp1 expression via both PKC-and MAPK-dependent pathways, activation of NF-kappaB by ET-1 is mediated by a PKCepsilon/reactive oxygen species (ROS) cascade. Taken together, these results suggest that by activating NF-kappaB via PKCepsilon/ROS cascade and increasing Sp1 expression through both PKCepsilon- and MAPK-dependent pathways, ET-1 may activate Glut1 transcription by enhancing interaction between nuclear NF-kappaB and Sp1 as well as their binding to enhancer 2.

Endothelin-1 induction of Glut1 transcription in 3T3-L1 adipocytes involves distinct PKCepsilon- and p42/p44 MAPK-dependent pathways.

We have shown previously that chronic exposure to endothelin-1 (ET-1) may stimulate GLUT1-mediated glucose transport in 3T3-L1 adipocytes via both protein kinase C (PKC)- and mitogen-activated protein kinase (p42/p44 MAPK)-dependent pathways. In the present study, by using a luciferase reporter driven by Glut1 promoter and enhancers (pLuc-GT1/E1/E2) and various constitutively active and dominant negative mutants of PKC isoforms, we identified PKCepsilon as the PKC isoform involved. In addition, we provide evidence that there is no direct interaction between ET-1 activated PKCepsilon and MAPK, at least at the kinase activity level. Furthermore, investigations employing deletion mutants of pLuc-GT1/E1/E2 to locate the putative ET-1 responsive sites and inhibitory agents to suppress the activities of putative transcription factors suggested that transcription factors CREB, Sp1 and NF-kappaB were involved. In summary, the results of this study indicate that ET-1 induction of Glut1 transcription involves distinct PKCepsilon- and MAPK-dependent pathways, as well as downstream transcription factors CREB, Sp1 and NF-kappaB.

Pretreatment with muscarinic receptor agonist activates a calcium-inhibitable adenylate cyclase in GH3 pituitary cells.

We have demonstrated previously that pretreatment of GH3 pituitary cells with muscarinic agonists may induce a higher cAMP formation in response to vasoactive intestinal peptide (VIP) or forskolin. In the present study, we further examined the adenylate cyclase (AC) that may be involved. We found that carbachol-pretreatment enhanced both VIP- and forskolin-activated AC activities. The addition of calcium ions to the incubation buffer diminished this enhancing effect. Carbachol was found to induce a decrease in intracellular calcium concentration [Ca2+]i by inhibiting calcium influx through L-type Ca2+ channels. However, the incubation of cells in Ca(2+)-free buffer or in the presence of L-type Ca2+ channel blockers had no influence on forskolin-stimulated cAMP formation, although both treatments induced decreases in [Ca2+]i as carbachol did. On the other hand, incubation in the presence of LaCl3 at a low concentration not being able to enter cells, forskolin-stimulated cAMP formation as well as the enhancing effect of carbachol-pretreatment on this response, were both suppressed. Similar phenomena were observed when membrane-bound AC activities were measured in the presence of LaCl3. Taken together, these results seem to suggest that pretreatment of GH3 cells with muscarinic receptor agonist may activate a Ca(2+)-inhibitable AC for a higher stimulated response. Low intracellular calcium concentrations are essential but not sufficient for this effect.

Synergistic effect of prostaglandin F2alpha and cyclic AMP on glucose transport in 3T3-L1 adipocytes.

The combined effect of prostaglandin F2alpha (PGF2alpha) and cAMP on glucose transport in 3T3-L1 adipocytes was examined. In cells pretreated with PGF2alpha and 8-bromo cAMP for 8 h, a synergy between these two agents on glucose uptake was found. Insulin-stimulated glucose transport, on the other hand, was only slightly affected. The synergistic effect of these two agents was suppressed in the presence of cycloheximide and actinomycin D. In concord, immunoblot and Northern blot analyses revealed that GLUT1 protein and mRNA levels were both increased in cells pretreated with both PGF2alpha and 8-bromo cAMP, greater than the additive effect of each agent alone. The synergistic action of PGF2alpha with 8-bromo cAMP to enhance glucose transport was inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor. In addition, in cells depleted of diacylglycerol-sensitive PKC by prolonged treatment with 4beta-phorbol 12beta-myristate 13alpha-acetate, a PKC activator, the synergistic effects of PGF2alpha and 8-bromo cAMP on glucose transport and GLUT1 mRNA accumulation were both abolished. Taken together, these results indicate that PGF2alpha may act with cAMP in a synergistic way to increase glucose transport, probably through enhanced GLUT1 expression by a PKC-dependent mechanism.

Synergistic effect of endothelin-1 and cyclic AMP on glucose transport in 3T3-L1 adipocytes.

We have demonstrated previously that chronic exposure to endothlin-1 enhances glucose transport in 3T3-L1 adipocytes via augmented GLUT1 mRNA and protein accumulation. In the present study, we further examined the combined effect of endothelin-1 (ET-1) and cAMP on glucose transport. In cells pretreated with ET-1 and 8-bromo cAMP for 8 h, a synergy between these two agents on glucose uptake was found. Insulin-stimulated glucose transport, on the other hand, was only slightly affected. The synergistic effect of these two agents was suppressed in the presence of cycloheximide and actinomycin D. Immunoblot and Northern blot analyses revealed that GLUT1 protein and mRNA levels were both increased in cells pretreated with both ET-1 and 8-bromo cAMP, greater than the additive effect of each agent alone. Further examination demonstrated that the stability of GLUT1 mRNA was markedly enhanced in the presence of both ET-1 and cAMP. To investigate the transcriptional activation of Glut1 gene, transient transfection of cells with luciferase reporter construct driven by Glut1 promoter was performed. We found that Glut1 transcription was also increased by ET-1 and cAMP in a synergistic fashion. In addition, similar synergy between ET-1 and beta-adrenergic agonists on glucose transport was found. The synergistic action of ET-1 with 8-bromo cAMP to enhance glucose transport was inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor, and was mimicked by 4beta-phorbol 12beta-myristate 13alpha-acetate (PMA), a PKC activator. Furthermore, PMA was found to act synergistically with 8-bromo cAMP to induce Glut1 transcription and ET-1 was shown to activate novel PKCdelta and PKC. Taken together, these results indicate that ET-1 may act with cAMP in a synergistic way to increase glucose transport, probably through enhanced GLUT1 expression via a PKC-dependent mechanism.

Thapsigargin and EGTA inhibit endothelin-1-induced glucose transport.

We have previously demonstrated that ET-1 may enhance glucose transport in 3T3-L1 adipocytes, secondarily to its stimulatory effect on GLUT1 gene expression by a mitogen-activated protein kinase (MAPK)-dependent pathway. In the present study, we further tested the involvement of Ca(2+) in glucose uptake in response to ET-1. Among a variety of Ca(2+)-related agents tested, EGTA and thapsigargin were found to suppress both the glucose uptake and intracellular Ca(2+) mobilization induced by ET-1, as determined by Fura-2 analysis. However, a phospholipase C inhibitor, U73122, also eliminated the intracellular calcium mobilization induced by ET-1, but had no effect on ET-1-stimulated glucose uptake. The finding that neither EGTA nor thapsigargin had any influence on ET-1-induced MAPK activation implies that some mechanism downstream of MAPK activation is involved. Further investigation showed that both agents exerted global inhibitory effects on protein and RNA syntheses. Since both thapsigargin and EGTA may deplete endoplasmic reticulum (ER) Ca(2+) stores, our results suggest that (1) ET-1-induced glucose transport is independent of ET-1's effect on Ca(2+) mobilization and (2) depletion of ER Ca(2+) stores per se may interfere with ET-1's effect on GLUT1 expression.

Prostaglandin F2alpha increases glucose transport in 3T3-L1 adipocytes through enhanced GLUT1 expression by a protein kinase C-dependent pathway.

The effect of prostaglandin F2alpha (PGF2alpha) on glucose transport in differentiated 3T3-L1 adipocytes was examined. Whereas PGF2alpha had little influence on insulin-stimulated 2-deoxyglucose uptake, it increased basal glucose uptake in a time- and dose-dependent manner, reaching maximum at approximately 8 h. The long-term effect of PGF2alpha on glucose transport was inhibited by both cycloheximide and actinomycin D. In concord, while the content of GLUT4 protein was not altered, immunoblot and Northern blot analyses revealed that both GLUT1 protein and mRNA levels were increased by exposure of cells to PGF2alpha. The effect of PGF2alpha on glucose uptake was inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor. In addition, in cells depleted of diacylglycerol-sensitive PKC by prolonged treatment with 4beta-phorbol 12beta-myristate 13alpha-acetate (PMA), the stimulatory effects of PGF2alpha on glucose transport and GLUT1 mRNA accumulation were both inhibited. In accord, PMA was shown to stimulate GLUT1 mRNA accumulation. To further investigate if PKC may be activated by PGF2alpha, we tested several diacylglycerol-sensitive PKC isozymes and found that PGF2alpha was able to activate PKCepsilon. Taken together, these results indicate that PGF2alpha may enhance glucose transport in 3T3-L1 adipocytes by stimulating GLUT1 expression via a PKC-dependent mechanism.

Differential effects of endothelin-1 on calcium mobility in cultured porcine pigment epithelial cells of iris and retina.

BACKGROUND AND PURPOSE: Photoreceptor function depends on an intact retina pigment epithelial (RPE) cell layer. Transplantation of iris pigment epithelial (IPE) cells to the subretinal space offers a potential alternative to RPE transplantation in patients with severe retinal diseases such as macular degeneration. Whether the functions of IPE and RPE are similar or different is not well established. The purpose of this study was to identify the characteristics of IPE cells and RPE cells by detecting the effects of endothelin-1 (ET-1) on intracellular free Ca(2+) ([Ca(2+)](i)) mobility in these cells. METHODS: Iris and retina pigment epithelial cells were cultured from porcine eyeballs. After the cells were loaded with fura-2/AM, fluorescence was monitored with a fluorescent spectrophotometer by continuously recording excitation signals at 340 nm and 380 nm and emission signals at 510 nm. RESULTS: IPE and RPE cells induced a significant increase in [Ca(2+)](i) under exposure to 10(-7), 10(-8), and 10(-9) mol/L ET-1 in Ca(2+)-containing buffer. In the presence of Ca(2+)-free buffer, ET-1 alone induced an increase in [Ca(2+)](i) in iris but not in retina pigment epithelial cells. In Ca(2+)-free buffer, pretreatment of the IPE cells with 10(-7) mol/L ET-1 partially inhibited thapsigargin-induced [Ca(2+)](i) increase and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced Ca(2+) release. Similarly, pretreatment of the IPE cells with thapsigargin or CCCP also partially inhibited ET-1-induced [Ca(2+)](i) increase. The [Ca(2+)](i) release induced by 10(-7) mol/L ET-1 was not inhibited by the phospholipase C inhibitor U73122. In Ca(2+)-containing buffer, the ET(A) receptor antagonist BQ123 and ET(B) receptor antagonist BQ788 partially inhibited 10(-7) mol/L ET-1-induced [Ca(2+)](i) increase in IPE cells. However, BQ123 partially inhibited the 10(-7) mol/L ET-1-induced [Ca(2+)](i) increase in RPE cells. Nifedipine partially inhibited 10(-7) mol/L ET-1-induced [Ca(2+)](i) increase in both types of pigment cells. CONCLUSIONS: These results suggest that iris and retina pigment epithelial cells possess distinguishing characteristics because the ET-1-induced [Ca(2+)](i) increases in these 2 types of pigment epithelial cells are regulated by distinct mechanisms.

Effects of commercial antiglaucoma drugs to glutamate-induced [Ca2+)]i increase in cultured neuroblastoma cells.

Over releasing of glutamate and cellular calcium influx always results in neuronal death. In the present study, we investigated various commercial antiglaucoma drugs including timolol (0.58 microM to 58 microM), betaxolol (1.62 microM to 162 microM), carteolol (6.8 microM to 680 microM), pilocarpine (4.08 microM to 408 microM), latanoprost (0.01 microM to 1.1 microM), dorzolamide (6.16 microM to 616 microM), brinzolamide (2.6 microM to 260 microM), brimonidine (0.68 microM to 68 microM), dipivefrin (0.28 microM to 28 microM) and preservative benzalkonium chloride on their effects to inhibit glutamate-induced intracellular free Ca(2+) ([Ca(2+)](i)) increase in cultured N1E-115 neuroblastoma cells. These drugs were diluted from original concentrations to 1/100, 1/1000 and 1/10000. The [Ca(2+)](i) mobility was studied after loading with fura-2-AM and analyzed by spectrofluorometry. It was found that betaxolol, dipivefrin and brimonidine have remarkable effects not only to inhibit the glutamate-induced [Ca(2+)](i) increase but also to decrease the basal [Ca(2+)](i). In the case of other drugs, only high concentration of timolol (58 microM) exhibited significant effect to completely prevent glutamate-induced [Ca(2+)](i) increase. Moreover, benzalkonium chloride did not exhibit any inhibitive effect. These results indicate that betaxolol, dipivefrin and brimonidine may have neuroprotective effects to inhibit the glutamate-induced over Ca(2+) influx damage.

Change of cytosolic Ca(2+) mobility in cultured bovine corneal endothelial cells by endothelin-1.

The effect of endothelin-1 (ET-1) on the intracellular free Ca(2+) ([Ca(2+)](i)) in cultured bovine corneal endothelial cells was studied after loading with fura-2-AM. In Ca(2+)-containing buffer and Ca(2+)-free buffer, ET-1 induced a significant rise in [Ca(2+)](i) at concentrations from 10(-9) to 10(-7) M. In Ca(2+)-free buffer, pretreatment of the cells with ET-1 inhibited thapsigargin-induced [Ca(2+)](i) increase and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced Ca(2+) release by 99% and 62%, respectively. Pretreatment of the cells with thapsigargin or CCCP also inhibited ET-1-induced [Ca(2+)](i) rise by 36% and 92%, respectively. In Ca(2+)-containing buffer, the ET(A) receptor antagonist (BQ123) and ET(B) receptor antagonist (BQ788) partially inhibited ET-1-induced [Ca(2+)](i) by 92% and 98%, respectively. Nifedipine and La(3+) also inhibited ET-1-induced [Ca(2+)](i) increase by 26% and 91%, respectively. The intracellular calcium release caused by ET-1 was partially inhibited by phospholipase C inhibitor (U73122). After incubation of the cells with ET-1 in Ca(2+)-free buffer, the addition of 5 mM CaCl(2) increased Ca(2+) influx, implying that release of Ca(2+) from internal stores caused by ET-1 further induced capacitative Ca(2+) entry. These data suggest that ET-1-induced [Ca(2+)](i) rise in bovine corneal endothelial cells are mediated by ET(A) receptor, ET(B) receptor, La(3+)-sensitive Ca(2+) pump and L-type voltage-operated Ca(2+) channel, leading to Ca(2+) influx. ET-1 also increased the internal Ca(2+) release from endoplasmic reticulum and mitochondria Ca(2+) stores followed by capacitative Ca(2+) entry. ET-1-induced intracellular Ca(2+) release was modulated by phospholipase C-coupled events.

Mechanism of inhibition of insulin-stimulated glucose transport by 4-bromocrotonic acid in 3T3-L1 adipocytes.

We have demonstrated previously that 4-bromocrotonic acid (Br-C4) inhibited insulin-stimulated glucose transport by interfering with GLUT4 translocation. In the present study, we further examined the underlying mechanism involved. Since insulin-induced insulin receptor substrate-1-associated phosphatidylinositol (PI) 3-kinase activity was not altered by Br-C4, we determined and found insulin activation of protein kinase B (PKB) and protein kinase Clambda (PKClambda) were both inhibited. However, time-course studies showed that only the inhibition of PKB activation correlated with the inhibition of insulin-stimulated glucose transport. In concert, insulin-stimulated Ser(473/474) phosphorylation on PKB(alpha/beta) were similarly decreased by Br-C4. The finding that okadaic acid-stimulated glucose transport and PKClambda activity were both inhibited by Br-C4 suggested that the effect of Br-C4 on Ser(473/474) phosphorylation was not mediated by protein phosphatase 2A. Moreover, whereas Br-C4 nearly abolished insulin-stimulated integrin-linked kinase (ILK) activity, it only inhibited insulin-stimulated PKB activity by 20%, implying that ILK was not the major kinase for Ser(473/474) phosphorylation. Taken together, these results support the notion that PKB is involved in insulin-stimulated glucose transport. In addition, Br-C4 seems to inhibit insulin-stimulated glucose transport via inhibiting insulin activation of PKB, probably by interfering with insulin activation of an upstream kinase responsible for the phosphorylation of Ser(473/474) residue.