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L Y Chau

Publications and source records attributed to L Y Chau.

At least 19 recordsLinked to original sources
PubMed2001

Adenovirus-mediated heme oxygenase-1 gene transfer inhibits the development of atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Increasing evidence supports the role of heme oxygenase-1 (HO-1) in cytoprotective response and iron homeostasis. The object of this study was to investigate whether adenovirus-mediated gene transfer of HO-1 in arteries reduces iron overload and inhibits lesion formation in apolipoprotein E (apoE)-deficient mice. METHODS AND RESULTS: Infection of rat aortic smooth muscle cells with adenovirus carrying the human HO-1 gene (Adv-HO-1) resulted in a high-level expression of HO-1 protein, which effectively reduced the hemin-induced iron overload in these cells. Adenovirus-mediated gene transfer in arteries in vivo was achieved by direct injection of Adv-HO-1 into the left ventricles of anesthetized animals. Transgene was expressed in the endothelium and aortic lesion of apoE-deficient mice after they had received recombinant adenovirus for 1 week and gradually decayed during the next 5 weeks. When young apoE-deficient mice (14 weeks old) received Adv-HO-1 (2.5 x 10(9) pfu) for 6 weeks, lesions that developed in the aortic root or aortic arch were significantly smaller than those in control littermates receiving empty viral vector. Furthermore, the iron deposition as well as tissue iron content was much less in aortic tissue of Adv-HO-1-treated mice. The inhibitory effect of HO-1 gene transfer on the progression of advanced lesions was also observed in older apoE-deficient mice (20 weeks old) receiving Adv-HO-1 intraventricularly. CONCLUSIONS: Overexpression of HO-1 in vascular cells facilitates iron metabolism and attenuates development of atherosclerosis in apoE-deficient mice.

Adenoviridae↗
PubMed2000

Differential regulation of H- and L-ferritin messenger RNA subunits, ferritin protein and iron following focal cerebral ischemia-reperfusion.

Iron may catalyse the production of reactive oxygen species during post-ischemic reoxygenation and subsequently lead to brain damage. Ferritin, an iron sequestering and storage protein, can also be a source of iron after ischemic insult. However, its role in ischemia-reperfusion has not been carefully investigated. In the present study, we examined the temporal and spatial induction profiles of both H- and L-ferritin messenger RNA and protein in a well-defined focal cerebral ischemia model. Results of northern blot analysis showed a delayed and prolonged induction of both H- and L-ferritin messenger RNA in the ischemic cortex of rats subjected to 60min ischemic insult. A significant induction of both H- and L-ferritin messenger RNA was observed at 12h and remained elevated for up to 336h after the onset of reperfusion. At the peak level, quantitative analysis of the blot indicated a 2.5-fold and a six-fold increase in H- and L-ferritin messenger RNA, respectively, compared with the sham-operated controls. No apparent change in the levels of either messenger RNA was observed in the contralateral side. Results of in situ hybridization studies revealed constitutive expression of both H- and L-ferritin messenger RNA throughout the brain in sham-operated animals, in particular the hippocampus and the piriform cortex. Nevertheless, the signal intensity of H-ferritin messenger RNA was much higher than that of L-ferritin messenger RNA. Seventy-two hours after 60min ischemia, marked expression of H-ferritin messenger RNA was observed in the area surrounding the middle cerebral artery irrigated cortex, the medial part of the caudoputamen and in the subfield of the CA1 hippocampal region of the ipsilateral hemisphere. Similarly, a large induction of L-ferritin messenger RNA was also noted in several areas, including the middle cerebral artery irrigated cortex, the lateral part of the caudoputamen and the stratum pyramidale of the CA1 hippocampal region, which were totally different from areas where H-ferritin messenger RNA was found. At 336h after ischemia, increased expression of H-ferritin messenger RNA was observed in the peri-necrosis and ipsilateral thalamus regions, while L-ferritin messenger RNA was noted exclusively at the edge within the necrosis. Results of immunohistochemical study further revealed that ferritin immunoreactivity was present in the same areas where increased ferritin messenger RNA was found. Sixty-minute ischemia also led to iron deposition in discrete areas. Iron deposition was highly associated with the induction of ferritin, particularly in the macrophage- and microglia-positive areas where cell death or tissue necrosis was noted.In summary, our initial findings indicate that ischemic insult leads to induction of both H- and L-ferritin messenger RNA. In the present study, although the temporal induction profiles were similar, the major expression areas for these two genes were totally different. Ferritin immunoreactivity was observed in the same areas where increased ferritin messenger RNA was found. Ischemia also resulted in iron deposition, which highly associated with the ferritin immunoreactivity. The exact regulatory mechanism and pathological significance for the differential expression of H- and L-ferritin genes following ischemia/reperfusion remain to be clarified.

Animals↗
PubMed2000

Iron and atherosclerosis.

Iron is a vital element in life. However, it may participate in diverse pathological processes by catalyzing the formation of reactive oxygen free radicals. During the past decade, considerable evidence has supported the role of oxidative stress in the development of atherosclerosis and related cardiovascular diseases. The oxidation of low-density lipoprotein (LDL) and lipid is believed to be one of the crucial events leading to plaque formation in vasculature. It has been hypothesized that iron-mediated oxidation is involved in this process. In favor of this idea, several epidemiological studies have shown that the level of body iron stores is positively correlated with the incidence of coronary heart disease in human populations. However, some studies have yielded conflicting results. Recently, studies conducted in our laboratory and others have demonstrated that iron deposition is prominent in human atherosclerotic lesions. The iron deposits appear to colocalize with ceroid, which is an end product of extensively oxidized lipid and protein complex, in lesions, providing histological evidence to support the iron hypothesis. Additional experiments in animals have further revealed that the severity of atherosclerosis can be markedly influenced by iron overload or deficiency. Collectively, these data provide a strong pathological basis to support the detrimental role of iron in vascular damage and progression of the disease.

Animals↗
PubMed1999

Post-transcriptional regulation of H-ferritin mRNA. Identification of a pyrimidine-rich sequence in the 3'-untranslated region associated with message stability in human monocytic THP-1 cells.

We have previously demonstrated that phorbol myristate acetate (PMA) up-regulates H-ferritin gene expression in myeloid cells by stabilization of its message. In the present report, we showed that insertion of the 3'-untranslated region (3'-UTR) of H-ferritin mRNA at the 3'-end of luciferase coding sequence significantly reduced the stability of luciferase mRNA in human monocytic THP-1 cells. However, the half-life of the chimeric transcript was markedly prolonged after PMA treatment. A cytosolic protein factor from THP-1 cells was found to specifically bind to H-ferritin 3'-UTR. PMA treatment of THP-1 cells resulted in the reduction of the RNA binding activity in a time-dependent manner. Deletion analysis and RNase T1 mapping revealed a pyrimidine-rich sequence within the 3'-UTR which interacts with the protein factor. Competition experiments with homoribopolymers further demonstrated the importance of uridines for the binding activity. Point mutations in uridines of the pyrimidine-rich sequence reduced the protein binding to 3'-UTR, while increasing the stability of the chimeric luciferase transcript. Together, these results demonstrate that the pyrimidine-rich sequence in the 3'-UTR is involved in post-transcriptional regulation of H-ferritin gene expression in myeloid cells.

3' Untranslated Regions↗
PubMed1999

Erythrophagocytosis and iron deposition in atherosclerotic lesions.

Iron deposition has been shown to be prominent in atherosclerotic lesions. However, the source of iron accumulated in arterial walls is unclear. In present report, we provide the histological evidence to demonstrate the localization of erythrocytes in atherosclerotic lesions from experimental animals. As revealed by scanning and transmission electron microscopy, the circulating erythrocytes were found to be present in intima of atherosclerotic aortas from apoE-deficient mice. These erythrocytes appeared to be readily phagocytosed by macrophages in lesions. The erythrophagocytosis was also evident in lesions from cholesterol-fed rabbits. Furthermore, the iron deposition was detectable in the region with erythrocytes. When the aortic sections of humans and apoE-deficient mice were immunostained with specific antibody to hemoglobin, it was clearly shown that the positive stain was detectable in macrophage-derived foam cells. Immunostaining of serial sections with specific antibodies to heme oxygenase-1 (HO-1) and ferritin further demonstrated the colocalization of HO-1 and ferritin in area with positive immunoreactivity for hemoglobin. Likewise, Perls' reaction revealed the positive iron stain in the same region. Collectively, these results suggest that hemoglobin/heme released from the phagocytosed erythrocytes may contribute to at least part of iron deposited in atherosclerotic lesions.

Animals↗
PubMed1999

Iron-deficient diet reduces atherosclerotic lesions in apoE-deficient mice.

BACKGROUND: Iron deposition is evident in human atherosclerotic lesions, suggesting that iron may play a role in the development of atherosclerosis. To test this idea, the correlation between the extent of iron deposition and the severity of atherosclerosis in apolipoprotein E (apoE)-deficient mice was investigated. Furthermore, the effect of a low-iron diet on the progression of atherosclerotic lesions in these animals was evaluated. METHODS AND RESULTS: Iron deposition in tissues of apoE-deficient mice was examined by Perls' staining method. The results clearly demonstrated that iron deposits are present in atherosclerotic lesions and tissue sections of heart and liver in an age-dependent manner. When the young mice received a low-iron diet for 3 months, the hematocrit, serum iron, hemoglobin, and cholesterol concentrations were not significantly altered compared with those of littermates placed on a chow diet. However, the serum ferritin level of animals in the iron-restricted group was 27% to 30% lower than that of the control group in either sex. Furthermore, the lipoproteins isolated from the iron-restricted group exhibited greater resistance to copper-induced oxidation. Histological examination revealed that atherosclerotic lesions developed in mice fed a low-iron diet were significantly smaller than those found in control littermates. Likewise, the iron deposition as well as tissue iron content was much less in aortic tissues of the iron-restricted animals. Circulating autoantibodies to oxidized LDL and immunostains for epitopes of malondialdehyde-modified LDL detected on lesions were also significantly lower in mice fed a low-iron diet. CONCLUSIONS: Iron deposition is closely associated with the progression of atherosclerosis in apoE-deficient mice. Restriction in dietary iron intake leads to significant inhibition of lesion formation in these animals. These results suggest that the beneficial effect of a low-iron diet may be mediated, at least in part, by the reduction of iron deposition as well as LDL oxidation in vascular lesions.

Alkaline Phosphatase↗
PubMed1999

Copper-induced apoptosis and immediate early gene expression in macrophages.

The death of macrophage-derived foam cells contributes to the formation of the lipid core in atherosclerotic lesions. Although the underlying mechanism is not yet clear, apoptosis has been shown to be responsible, at least in part, for the cell death of lipid-laden macrophages in atherosclerotic plaques. In the present study, we demonstrated that copper, in the presence of 8-hydroxyquinoline, was able to induce apoptosis of murine J774.A1 cells in culture. Ceruloplasmin exerts similar a effect, but not iron or hemin. Further experiments demonstrated that the expression of immediate early genes, including c-jun, c-fos and egr-1, was also induced by copper treatment in these cells, although only egr-1 mRNA was induced in a time- and dose-dependent manner. The antioxidant, N-acetylcysteine, exhibited remarkable inhibitory effect on the copper-induced apoptosis dose-dependently. Time course experiment revealed that prior treatment of cells with N-acetylcysteine is essential for the anti-apoptotic effect of this compound. Results also demonstrated that under the condition; in which N-acetylcysteine inhibited the copper-induced apoptosis, this antioxidant also abolished the gene expression of egr-1. Collectively, these results suggest that egr-1 gene expression is closely associated with the apoptosis induced by copper in macrophages.

Animals↗
PubMed1999

The role of interleukin 12 in the development of atherosclerosis in ApoE-deficient mice.

The cytokine profile of atherosclerotic aortas from apoE-deficient mice was assessed by reverse transcriptase-polymerase chain reaction. The results clearly showed that the expression of mRNA for IL-12p40 was evident in aortas from 3-month-old apoE-deficient mice. The mRNA for IL-10 was detected in aorta from these mice at the age of 6 months, indicating that expression of IL-12 is earlier than that of IL-10 in these animals. Concurrent with IL-12p40, the mRNA for the T-cell cytokine IFN-gamma, but not IL-4, was detected in aortas of mice at young and old ages. Both in situ hybridization and immunostaining further demonstrated the localization of IL-12 in macrophages of atherosclerotic lesions. Immunohistochemistry also demonstrated the expression of costimulatory molecules B7-1 and B7-2 in macrophages, suggesting that activation of T lymphocytes by macrophages may occur via surface antigens in lesions. When the immunoglobulin isotype of the antioxidized LDL antibodies in sera of apoE-deficient mice was determined, it revealed that both IgM and IgG were present. Furthermore, IgG2a is predominant and comprises approximately 50% of the antioxidized LDL IgG in sera from young mice (3 months), but decreased to lower levels (35%) in older mice (6 months). Daily administration of IL-12 led to an increase in serum levels of antioxidized LDL antibodies and accelerated atherosclerosis in young apoE-deficient mice compared with control mice injected with PBS alone. Taken together, these data suggest that IL-12 plays an active role in regulating the immune response during the early phase of atherosclerosis in apoE-deficient mice.

Animals↗
PubMed1998

Colocalization of iron and ceroid in human atherosclerotic lesions.

The presence of ceroid, a complex of protein associated with oxidized lipids, is commonly observed in human atherosclerotic lesions. When the human aortic walls were examined by Perls' staining, it was found that the iron deposits were evident in aortas with atherosclerosis. The extent of iron deposition was associated with the severity of the lesion. Furthermore, the iron deposits appeared to be colocalized with ceroids either extracellularly or intracellularly in foam cell-like macrophages or smooth muscle cells. Electron microscopy and X-ray microanalysis revealed that some of the extracellular iron aggregates were present within the ceroids. Likewise, some of the subcellular iron aggregates were found to be located near the lipid droplets or within the ceroids of foam cells. Collectively, these observations support the theory that the lipid oxidation occurring in lipid-laden cells of aortic lesions is facilitated by iron-overload in these cells.

Adult↗
PubMed1998

Expression of heme oxygenase-1 in atherosclerotic lesions.

Heme oxygenase-1 (HO-1) is a heme-degradation enzyme induced under various oxidative stress conditions. To elucidate the potential involvement of HO-1 in atherogenesis, the expression of this enzyme in atherosclerotic lesions of apolipoprotein E-deficient mice and humans were examined. Both immunostaining and in situ hybridization clearly demonstrated that the expression of HO-1 was prominent in endothelium and foam cells/macrophages of thickened intima in lesions from both humans and experimental animals. The expression of this enzyme was also detected in medial smooth muscle cells of advanced lesions. The induction of HO-1 mRNA was observed in murine peritoneal macrophages after treatment with oxidized low density lipoprotein (LDL) but not with native LDL in a dose-dependent manner. Time course study demonstrated that the induction was prominent at 3 hours, reached a maximal induction at 6 hours, and remained evident up to 24 hours after oxidized LDL treatment. The degree of induction was in concordant with the extent of oxidation in the LDL preparation. Lysophosphatidylcholine, one of the major components present in oxidized LDL, was ineffective to induce the gene expression, suggesting that other lipophilic substances derived from LDL oxidation are responsible for the induction of HO-1. These results clearly demonstrate that HO-1 is one of the stress proteins expressed in atherosclerotic lesions.

Aged↗
PubMed1997

Transcriptional regulation of platelet-activating factor receptor gene in B lymphoblastoid Ramos cells by TGF-beta.

The expression of platelet-activating factor (PAF) receptor gene was up-regulated in a time- and dose-dependent manner in a B cell line (Ramos) following exposure to TGF-beta 2. The TGF-beta 2-induced increment of PAF receptor mRNA was at least partly due to an increase in transcriptional rate as demonstrated by nuclear run-off experiments. Transient transfection of cells with PAF receptor transcript I gene promoter fused to a luciferase reporter gene revealed that the TGF-beta-responsive element (T beta RE) lies between the sequence from -44 to -17 relative to the transcriptional start site. Insertion of the T beta RE upstream of the unresponsive minimal thymidine kinase promoter conferred the TGF-beta-inducibility. Gel mobility shift assay demonstrated the specific binding of nuclear factors to the T beta RE. The T beta RE binding activity was gradually increased and reached a maximum at 3 h and subsequently returned to basal level at 5 h in cells following TGF-beta 2-treatment. Concomitant treatment of cells with cycloheximide abolished the increases in both T beta RE-binding activity and expression of PAF receptor mRNA, indicating that de novo protein synthesis is required to exert TGF-beta 2 effect. Methylation interference analysis revealed that the T beta RE-binding protein recognized a purine-rich sequence, 5'-GGGGTG-3'. Point mutations of the consecutive guanine nucleotides significantly reduced the DNA-binding activity and the TGF-beta-induced promoter activity. Collectively, these results clearly demonstrate that a T beta RE proximal to the transcriptional initiation site of the human PAF receptor transcript I gene mediates the up-regulation of PAF receptor gene expression in Ramos cells by TGF-beta 2.

B-Lymphocytes↗
PubMed1997

Analysis of the T cell receptor V beta repertoire in human aortic aneurysms.

Human aortic aneurysm is commonly characterized by the presence of advanced atherosclerosis associated with variable chronic adventitial inflammation. Histological examination of human aortic aneurysmal specimens revealed the presence of plasma cells and lymphoid aggregates in media and adventitia of the vessels. Immunostaining further demonstrated that CD3-positive T lymphocytes are present in follicles. Using a highly sensitive reverse transcription-polymerase chain reaction amplification method, the T cell receptor (TCR) V beta gene expression in aortic aneurysms was shown to be polyclonal. Furthermore. there was no preferential expression of any TCR V beta gene in the aortic tissue as compared with that in peripheral blood in aneurysmal patients. These results indicate that the TCR repertoire in aortic aneurysm is not restricted.

Aged↗
PubMed1996

Post-transcriptional regulation of H-ferritin gene expression in human monocytic THP-1 cells by protein kinase C.

The mRNA coding for H-ferritin was highly induced in human monocytic THP-1 cells following treatment with phorbol 12-myristate 13-acetate (PMA). The induction was detected at 3 h, reached maximal levels at 12 h, and was sustained for up to 48 h subsequent to PMA exposure. PMA-induced up-regulation of H-ferritin gene expression was also observed in other leukaemic cell lines, HL60 and U937, but not in non-leukaemic cell types, including human fibroblasts, endothelial cells and smooth muscle cells. The effect of PMA could be completely blocked by the protein kinase C inhibitor, H-7. Furthermore, treatment of THP-1 cells with bacterial phospholipase C also produced a marked increase in expression of H-ferritin mRNA, suggesting the activation of protein kinase C was responsible for the accumulation of mRNA. Nuclear run-off experiments demonstrated that PMA did not increase the transcriptional rate of the H-ferritin gene. In contrast, the half-life of the H-ferritin mRNA measured in the presence of actinomycin D was greatly prolonged in PMA-treated cells. The induction of H-ferritin mRNA by PMA required no protein synthesis. Conversely, treatment of THP-J cells with protein synthesis inhibitor, cycloheximide, resulted in a 4-5-fold increase in H-ferritin mRNA. The increase in the stability of the H-ferritin mRNA was also observed in cells treated with cycloheximide. Taken together, these results suggest that the stability of H-ferritin mRNA in THP-1 is subjected to regulation via a protein kinase C-mediated phosphorylation on existing putative protein factor(s).

Cell Differentiation↗
PubMed1996

Increased ferritin gene expression in atherosclerotic lesions.

To identify genes potentially implicated in atherogenesis, a cDNA library was constructed from human atherosclerotic aorta and differentially screened with 32P-labeled-cDNAs prepared from human normal and atherosclerotic aortas. Two cDNA clones exhibiting higher hybridization to the 32P-labeled cDNAs from atherosclerotic vessels were isolated and identified to be genes encoding L-ferritin and H-ferritin, respectively. Northern blot analysis confirmed that the expression of both ferritin genes was notably higher in human and rabbit atherosclerotic aortas than in their normal counterparts. A time-course study illustrated that both L- and H-ferritin mRNAs were markedly increased in aortas of rabbits after feeding with a high cholesterol diet for 6 wk, which was also the time period after which the formation of lesions became evident. In situ hybridization revealed that both L- and H-ferritin mRNAs were induced in endothelial cells and macrophages of human early lesions. The signals were also detected in the smooth muscle cells of advanced lesions. Immunostaining further identified the presence of ferritin protein in atherosclerotic lesions. On the other hand, Prussian blue stain revealed the presence of iron deposits in advanced lesions but not in early human or rabbit lesions. Further experiments with cultured human monocytic THP-1 cells and aortic smooth muscle cells demonstrated that ferritin mRNAs were subjected to up-regulation by treatment with IL-1 or TNF, while TGF, PDGF, and oxidized LDL did not affect the expression of either ferritin gene in both cell lines. Collectively, these results clearly demonstrate that ferritin genes are susceptible to induction in the course of plaque formation.

Animals↗
PubMed1995

Functional characterization of the promoter region of the platelet-activating factor receptor gene. Identification of an initiator element essential for gene expression in myeloid cells.

To understand the molecular mechanisms that direct the expression of the gene encoding the platelet-activating factor (PAF) receptor, the 5'-flanking region of the human PAF receptor gene was cloned, and its promoter activity in myeloid cell lines was characterized. By the 5'-rapid amplification of cDNA ends method and primer extension, the transcription initiation site was mapped to an adenosine residue 137 bases upstream of the ATG translation initiation codon. The promoter region lacks a typical TATA or CCAAT box. However, the sequence encompassing the transcription initiation site shows high homology to the initiator (Inr) sequence. Transfection of promonocytic U937 cells with recombinant plasmids containing a series of truncated segments of the 5'-flanking region linked to the luciferase reporter gene revealed that the sequence from nucleotides -44 to +27 relative to the transcription initiation site was sufficient to promote a high level of gene expression. The promoter activity was much lower in nonexpressing HeLa cells and promyelocytic HL-60 cells, which express relatively low levels of the PAF receptor. Gel mobility shift analysis demonstrated the binding of nuclear factors extracted from myelocytic cells to the -16/+18 sequence containing the Inr element. No binding activity was detected using the nuclear extracts from the nonmyelocytic HeLa cells. The DNA-protein complexes were sequence-specific since the binding was not significantly affected by the mutated Inr sequences or the Inr sequence of the terminal deoxynucleotidyltransferase gene. Furthermore, point mutations in the Inr element significantly reduced promoter activity in both U937 and THP-1 cell lines. When Me2SO or retinoic acid was used to induce granulocytic differentiation of HL-60 cells, a distinct Inr-protein complex was induced concurrently, but the complex was not observed in 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiated HL-60 cells or Me2SO-induced differentiated U937 cells, indicating that the inducible Inr binding activity is granulocyte-specific. These results suggest that distinct nuclear factors interact with the unique Inr element and play a role in the transcriptional regulation of the PAF receptor in various myeloid cells.

Base Sequence↗
PubMed1994

Agonist-induced down-regulation of platelet-activating factor receptor gene expression in U937 cells.

Prolonged exposure (8-24 h) of human promonocytic U937 cells to 100 nM 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (carbarmyl-PAF), a non-metabolizable analogue of platelet-activating factor (PAF), reduced the numbers of PAF receptors by 50-75%, as determined by the radioligand-binding assay. To clarify whether the down-regulation of receptor numbers is due to decreased expression level of the PAF-receptor gene, the effect of carbamyl-PAF on the steady-state level of PAF-receptor mRNA was examined by a highly sensitive reverse-transcriptase PCR method. A 50% decline in the level of PAF-receptor mRNA was observed in U937 cells pretreated with 100 nM carbamyl-PAF for 24 h. The effect of carbamyl-PAF was dose-dependent, with an EC50 value around 10 nM. PAF-receptor antagonist, SRI-63675, was able to attenuate the effect of carbamyl-PAF. Furthermore lysoPAF, at 1 uM, was unable to induce a significant decrease in PAF-receptor mRNA after incubation for 24 h, indicating that the effect of carbamyl-PAF was specific. The half-life of the PAF-receptor mRNA measured in the presence of actinomycin D was unaffected by carbamyl-PAF treatment. In contrast, nuclear run-off experiments demonstrated that the transcription rate of the PAF-receptor gene in carbamyl-PAF-treated cells was about 65% of that in control cells. These results suggest that the PAF receptor in U937 cells is subject to down-regulation by agonist, at least partly, at the transcriptional level.

Base Sequence↗
PubMed1993

Endothelin-mediated calcium response and inositol 1,4,5-trisphosphate release in neuroblastoma-glioma hybrid cells (NG108-15): cross talk with ATP and bradykinin.

Addition of endothelins (ETs) to neuroblastoma-glioma hybrid cells (NG108-15) induced increases in cytosolic free Ca2+ ([Ca2+]i) levels of labeled inositol monophosphates and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. The increases in [Ca2+]i elicited by the three ETs (ET-1, ET-2, and ET-3) were transient and did not show a sustained phase. Chelating extracellular Ca2+ in the medium by adding excess EGTA decreased the ET-mediated Ca2+ response by 40-50%. This result indicates that a substantial portion of the increase in [Ca2+]i was due to influx from an extracellular source. However, the increase in [Ca2+]i was not affected by verapamil or nifedipine (10(-5) M). A rank order potency of ET-1 > ET-2 > ET-3 is shown for the stimulated increase in [Ca2+]i, as well as labeled inositol phosphates, in these cells. ATP (10(-4) M) and bradykinin (10(-7) M) also induced the increases in [Ca2+]i and Ins(1,4,5)P3 in NG108-15 cells, albeit to a different extent. When compared at 10(-7) M, bradykinin elicited a five- to sixfold higher increase in the level of Ins(1,4,5)P3, but less than a twofold higher increase in [Ca2+]i than those induced by ET-1. Additive increases in both Ins(1,4,5)P3 and [Ca2+]i were observed when ET-1, ATP, and bradykinin were added to the cells in different combinations, suggesting that each receptor agonist is responsible for the hydrolysis of a pool of polyphosphoinositide within the membrane. ET-1 exhibited homologous desensitization of the Ca2+ response, but partial heterologous desensitization to the Ca2+ response elicited by ATP. On the contrary, ET-1 did not desensitize the response elicited by bradykinin, although bradykinin exhibited complete heterologous desensitization to the response elicited by ET-1. Taken together, these results illustrate that, in NG108-15 cells, a considerable amount of receptor cross talk occurs between ET and other receptors that transmit signals through the polyphosphoinositide pathway.

Adenosine Triphosphate↗