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Thrombin-stimulated elevation of human endothelial-cell cytoplasmic free calcium concentration causes prostacyclin production.

ABSTRACT: Endothelial cells are known to release prostacyclin (PGI2) in response to agonists, and this has generally been assumed to be caused, at least in part, by activation of a phospholipase A2 by elevated concentrations of cytoplasmic free calcium ([Ca2+]i). However, it has been shown in the blood platelet that agonists can cause arachidonate release without elevating [Ca2+]i. In the present study, rigorous analysis is made of the [Ca2+]i-dependence of PGI2 production in the human umbilical-vein endothelial cell. Thrombin caused a rapid increase in [Ca2+]i from the resting basal value of 0.1 microM to a peak, within 10-15 s, of approx. 2 microM. In the absence of extracellular Ca2+, [Ca2+]i then declined back to the resting value within 2-3 min. In the presence of extracellular Ca2+, [Ca2+]i partly decreased to a new steady-state value of approx. 1 microM. The elevated [Ca2+]i was maintained while the stimulus and the source of extracellular Ca2+ were present, suggesting that it was dependent on influx of Ca2+ across the plasma membrane. Thrombin stimulated the production of PGI2 in the presence or in the absence of extracellular Ca2+. However, the production of PGI2 was more prolonged in the presence of extracellular Ca2+. Total accumulated amounts of 6-oxo-prostaglandin F1 alpha on stimulation with thrombin without extracellular Ca2+ were only 65% of those accumulated with extracellular Ca2+ present. Cells depleted of extracellular and intracellular sources of Ca2+ by incubation with 1 mM extracellular EGTA and exposing them to ionomycin to discharge intracellular stores produced no elevation of [Ca2+]i on stimulation with thrombin or production of PGI2. The threshold [Ca2+]i required to support the production of PGI2 was measured to be 0.8-1.0 microM by using different doses of ionomycin selectively to increase [Ca2+]i. This relationship between [Ca2+]i and PGI2 production was similar to that produced by using different doses of thrombin. Our results show that the major and probably exclusive intracellular stimulus for the production of PGI2 by the vascular endothelial cell in response to thrombin is the elevation of [Ca2+]i.

SUBMITTER: Hallam TJ

PROVIDER: S-EPMC1148990 | biostudies-other | 1988 Apr

REPOSITORIES: biostudies-other

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Project description:Confluent human umbilical vein endothelial cells (HUVECs) were exposed to Thrombin (2 U/mL) for 2 hours. Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA). Four samples were obtained from these experiments, Control Monosome, Control Polysome, Thrombin Monosome, and Thrombin Polysome. Using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation. This calculation was made as follows: (thrombin polysome/thrombin monosome)/(control polysome/control monosome). Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study. TSAA data suggests that JunB is translationally regulated by thrombin stimulation. Immunocytochemistry, western blotting and RT-PCR were used to verify the results of TSAA. Polysome Profiling. HUVECs were stimulated with thrombin (2 U/mL) for 2 hours. Cycloheximide (CHX) was added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with cold HBSS containing CHX (100 ng/mL) and scraped from the dish and pelleted by centrifugation (2000 x g, 5 minutes). The supernatant was removed and cells were carefully resuspended in 375 microL Low Salt Buffer (LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3 minutes on ice. 125 microL LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2% Triton-X100) was then added and cells disrupted by pipetting. The mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4 degrees C) to remove nuclei and cellular debris. The supernatant was transferred to a new tube containing 50 microL LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 microL 5M NaCl. This mixture was then layered onto prepoured gradients (15-50% sucrose in LSB). Gradients were spun at 43,700 rpm for 90 minutes (4 degrees C) and then run on a density gradient flow cell fractionator (Isco) to obtain ribosomal profiles. Ribosomal fractions corresponding to monosomes (M) and polysomes (P) (Figure 1) were collected into Trizol LS (Invitrogen) and total RNA isolated according to the directions of the manufacturer. Total RNA was reverse transcribed using oligo dT primers in the presence of 33P dCTP to generate labeled cDNA probes from the M and P associated mRNA fractions of control and stimulated cells and these probes used to detect specific array elements on four identical Research Genetics (ResGen) Named Human Gene cDNA GeneFilters (GF 211, Invitrogen). These filters were washed using high stringency conditions and exposed to phosphorimaging screens for analysis using Pathways 3, proprietary software included with the ResGen filters. Hybridization signals were normalized to the average intensity of the hybridization signals from the particular array filter to correct for differing global hybridization efficiencies between filters. To determine if a particular hybridization signal was significantly above background levels, an initial screen of the array elements was conducted by dividing the hybridization signal of an individual cDNA spot by the background signal on particular filter. Background signal levels were determined using the Pathways 3 analysis software that measures the signal intensity of the area between cDNA spots and averages that signal over the area of the entire array. Array elements were considered to be distinguishable from background if the hybridization signal/background ratio was greater than 1.1. Signal intensities from each filter were normalized by dividing the raw signal intensity of the cDNA spot by the average signal intensity for the entire filter. The translation state (TS) of each qualifying array element was determined by dividing the normalized signal intensity of the element in the polysome fraction by the normalized signal intensity of the element in the monosome fraction: TS=P/M. The Translation Index (TLI), a measure of the change of translation states of individual genes, was determined by calculating the ratio of the treated cell TS to the control cell TS; TLI = TSe/TSc where e represents experimental or treated conditions and c represents controls. In this scenario, a gene that is not regulated by translation would have a TLI value of 1, actively translated genes would have a TLI value greater than 1, whereas a poorly translated or under-translated gene would have a TLI value less than 1. Arbitrary TLI values of 2.5 and 0.4 were used to categorize individual array elements as being translationally regulated. A measure of transcriptional activity was also determined from the signal intensities of the array elements. Summing the normalized signal intensities from the M and P fractions of the experimental cells and dividing that value by the sum of the normalized signal intensities from the control fractions calculated the Transcription Index (TCI). Therefore, TCI = (Me + Pe)/(Mc + Pc). If transcript levels for a specific gene do not change, the TCI value for that gene will be 1. Genes that are transcribed upon stimulation of the cells will have a TCI value greater than 1, while genes that are not transcribed or transcription is down-regulated upon stimulation will have a TCI value less than 1. Arbitrary values of 2 and 0.5, a doubling or halving of the amount of mRNA for that element, respectively, were used to categorize individual array elements as being transcriptionally regulated.

Project description:Three aspects of the calcium hypothesis we have proposed previously [Metcalfe, Pozzan, Smith & Hesketh (1980) Biochem. Soc. Symp. 45, 1-26] for the control of mitogenic stimulation of lymphocytes are examined in studies on the mitogenic action of the Ca2+ ionophore A23187 and its effect on cap formation. (1) Pig lymphocytes that were mitogenically stimulated by continuous incubation with 3H-labelled A23187 for 48 h contained between 3 and 15 amol of ionophore per cell. Lymphocytes exposed to 3H-labelled A23187 for 2h before washing the cells and resuspending them in ionophore-free medium were only stimulated mitogenically at 48h if the residual ionophore associated with the cells after washing was in the concentration range 3-15 amol per cell. When the cells were washed repeatedly after 2h incubation with ionophore to reduce the cell-associated ionophore below the critical concentration range, no mitogenic stimulation occurred as a result of short-term exposure to any ionophore concentration. Re-addition of ionophore to within the indicated range of cell-associated concentrations restored mitogenic stimulation at 48h. We conclude that large, short-term Ca2+ fluxes into the cells induced by the ionophore cannot generate a mitogenic signal that commits the cells to enter the cell cycle. (2) Further experiments with the ionophore showed that detectable mitogenic stimulation at 48h required a minimum of 3h exposure to optimal ionophore concentrations, and that maximal stimulation required at least 20h exposure. This is consistent with the view that a prolonged increase in the free cytoplasmic calcium concentration is required to stimulate the maximum proportion of the cells into the cell cycle. (3) Mouse splenic lymphocytes treated for short periods with very high ionophore concentrations (30 microM) in the presence of various external Ca2+ concentrations showed significant inhibition of cap formation of surface immunoglobulin receptors in the range 1-10 microM-Ca2+ in normal or depolarizing medium. We conclude that mitogens at optimal concentrations for the stimulation of lymphocytes do not cause any early increase in the free cytoplasmic Ca2+ concentration above 10 microM.

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Thrombin-stimulated elevation of human endothelial-cell cytoplasmic free calcium concentration causes prostacyclin production.

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