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Biomedical subjects

C J He

Publications and source records attributed to C J He.

33 records · Page 2Linked to original sources
PubMed1992

Transcriptional activation of the urokinase receptor gene by endothelin-1.

Using an immortalized human glomerular epithelial cell line (E71 A1), we studied the effect of endothelin-1 (ET-1), a potent vasoconstrictor peptide, on the synthesis of urokinase type plasminogen activator (u-PA) and its receptor (u-PAR). The results show that ET-1 had no effect on u-PA synthesis but induced an increase in u-PAR number (2.8 +/- 0.6 x 10(4) vs 1.2 +/- 0.5 x 10(4) sites per cell, p less than 0.001) without change in receptor affinity (280 +/- 80 pM vs 250 +/- 50 pM, NS), maximal effect being observed at 10(-7) M. Time course shows that a plateau was reached after a 24 hour incubation. ET-1 induced-increase in binding capacity was abolished by cycloheximide. ET-1 also induced an increase in u-PAR mRNA level, which was completely blocked by alpha-amanitin (5 micrograms/ml). Cycloheximide (1 microgram/ml) alone induced an increase in u-PAR mRNA level and this effect was enhanced when cycloheximide was combined with ET-1. Our data show that ET-1 can induce an increase in membrane expression of u-PAR through activation of the transcription of the u-PAR gene and de novo protein synthesis.

Amanitins↗
PubMed1992

Thrombin signal transduction mechanisms in human glomerular epithelial cells.

We have previously shown that alpha-thrombin exerted a mitogenic effect on human glomerular epithelial cells and stimulated the synthesis of urokinase-type (u-PA) and tissue-type plasminogen activator (t-PA) and of their inhibitor, plasminogen activator inhibitor 1 (PAI-1). In the present study, we investigate the signal transduction mechanisms of thrombin in these cultured cells. Thrombin induced an increase in intracellular free calcium concentrations ([Ca2+]i) in a dose-dependent manner, a plateau being reached at 1 U/ml thrombin. A 60% inhibition of this effect was produced by 300 nM nicardipine, a dihydroperidine agent, or by 4 mM EGTA, indicating that increase in [Ca2+]i was due in part to extracellular Ca2+ entry through L-type voltage-sensitive calcium channels. Thrombin also induced an increase in inositol trisphosphate (IP3), suggesting that phospholipase C activation and phosphatidylinositides breakdown were stimulated. Interestingly thrombin-stimulated cell proliferation measured by 3H thymidine incorporation was inhibited by 300 nM nicardipine, and restored by addition of 10(-8) M ionomycin, indicating that calcium entry was critical for the mitogenic signal of thrombin. Conversely, nicardipine did not modify thrombin-stimulated synthesis of u-PA, t-PA, and PAI-1. Both thrombin-stimulated cell proliferation and protein synthesis required protein kinase C activation since these effects were blocked by 10 microM H7, an inhibitor of protein kinases, and by desensitization of protein kinase C by phorbol ester pretreatment of the cells. Interestingly, DFP-inactivated thrombin which binds the thrombin receptor and gamma-thrombin, which has some enzymatic activity but does not bind to thrombin receptor, had no effect when used alone. Simultaneous addition of these two thrombin derivatives had no effect on [Ca2+]i, and 3H thymidine incorporation but stimulated u-PA, t-PA, and PAI-1 synthesis although to a lesser extent than alpha-thrombin. This effect also required protein kinase C activation to occur, presumably by a pathway distinct from phosphoinositoside turnover since it was not associated with IP3 generation. In conclusion, multiple signalling pathways can be activated by alpha-thrombin in glomerular epithelial cells: 1) Ca2+ influx through a dihydroperidine-sensitive calcium channel, which seems critical for mitogenesis; 2) protein kinase C activation by phosphoinositide breakdown, which stimulates both mitogenesis and synthesis of u-PA, t-PA, and PAI-1; 3) protein kinase C activation by other phospholipid breakdown can stimulate u-PA, t-PA, and PAI-1 synthesis but not mitogenesis.

Calcium↗
PubMed1992

Quantification and modulation of thrombomodulin activity in isolated rat and human glomeruli.

Thrombomodulin (TM), the endothelial cell surface receptor for thrombin-mediated activation of protein C and of its anticoagulant system, is involved in maintaining vascular nonthrombogenicity, and depressed TM activity may induce intravascular fibrin formation. TM antigen was previously found by immunohistochemical methods in rabbit glomeruli. We therefore attempted to identify the corresponding TM activity in isolated detergent-solubilized rat and human glomeruli. Like purified lung TM, rat glomeruli extracts accelerated the hydrolysis by activated protein C of the chromogenic substrate S-2238 in the presence of 10 nM thrombin, as determined by spectrophotometry. One mg glomerular protein promoted the formation of 681 +/- 115 nmol activated protein C, the equivalent of the amount generated by 845 ng of purified rabbit TM. TM activity correlated with the protein content of the glomerular extracts (r = 0.94). These extracts prolonged rat plasma activated partial thromboplastin time. Incubation of glomeruli with tumor necrosis factor-alpha (TNF) or E. coli lipopolysaccharide depressed their TM-like activity in a dose and time dependent manner. Incubation with TNF suppressed their anticoagulant activity. In human glomeruli, TM activity was also found at a level which corresponded to their TM antigen content, and was determined by ELISA with mouse monoclonal antibody. These results indicate that measurement of glomerular TM activity might help to clarify the mechanisms of intraglomerular fibrin deposition in renal diseases.

Animals↗
PubMed1992

Tumor necrosis factor alpha increases antifibrinolytic activity of cultured human mesangial cells.

Tumor necrosis factor alpha (TNF alpha) is likely to exert a major influence in the pathogenesis of glomerulopathies. Besides its proinflammatory properties. TNF alpha interacts with cell growth and synthesis of components of the fibrinolytic system. In this study, we report the effects of recombinant human TNF alpha on the synthesis of tissue-type plasminogen activator (t-PA) and its inhibitor (PAI-1) by human mesangial cells in culture. We first demonstrate that TNF alpha binds specifically to a single class of high affinity receptors (Kd 5.10(-11) M; 1500 receptors/cell). TNF alpha has an antimitogenic effect on human mesangial cells since it decreased DNA synthesis, measured by 3H-thymidine incorporation, in a dose-dependent manner. Release of cytosolic LDH and incorporated 51Cr was not increased by 100 ng/ml TNF alpha as compared with control, indicating that this monokine is not cytotoxic for cultured human mesangial cells. Zymographic analysis and reverse fibrin autography disclosed a 120 kD t-PA-PAI-1 complex and a 50 kD free form of PAI-1 in the supernatants of both unstimulated and TNF-stimulated cells; PAI-1 was released in excess and free t-PA was not observed. TNF alpha (0 to 100 ng/ml) had no effect on t-PA synthesis, but enhanced PAI-1 release in a time- and dose-dependent manner (97% increase of PAI-1 synthesis after a 24 hour incubation). This effect was abolished by cycloheximide, suggesting that protein synthesis was required. Northern blot analysis showed that TNF alpha increased the steady-state PAI-1 mRNA levels in a time-dependent manner, with a maximal effect at two hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1992

Functional endothelin 1 receptors on human glomerular podocytes and mesangial cells.

To determine if endothelin 1 (Et1) receptors are present in human glomeruli, and which glomerular cells possess these receptors, 125I Et1 binding to isolated glomeruli and cultured glomerular mesangial and epithelial cells was studied. The latter were identified as podocytes. We demonstrated that Et1 binds specifically and reversibly to isolated human glomeruli and to cultured glomerular mesangial and epithelial cells. Scatchard analysis of competitive inhibition of 125I Et1 binding gave the following results (m +/- SEM, n = 3): isolated glomeruli, Kd = 4.2 +/- 2.1 x 10(-10) M, Bmax = 8.1 +/- 1.2 x 10(10) sites/mg protein; mesangial cells, Kd = 5.2 +/- 1.5 x 10(-10) M, Bmax = 1.87 +/- 0.49 x 10(4) sites/cell; epithelial cells, Kd = 7.2 +/- 1.5 x 10(-10) M, Bmax = 2.46 +/- 0.15 x 10(4) sites/cell. These receptors seem to be functional, since in both mesangial and epithelial cells Et1 induces a rapid and transient increase in intracellular [Ca2+]i. All these results indicate that Et1 may regulate glomerular filtration rate through an autocrine-paracrine pathway on mesangial cells and on podocytes.

Calcium↗
PubMed1992

Intact Leaves Exhibit a Climacteric-Like Rise in Ethylene Production before Abscission.

The rate of ethylene production by intact, attached leaves of cotton plants (Gossypium hirsutum L.) during aging and senescence was studied using a continuous flow system that allowed air around enclosed leaves to be scrubbed to collect and assay ethylene. Senescence of lower leaves began around 150 d after planting in a controlled environment room. A progressive decline in the ethylene production rate was observed when comparing the 3rd, 6th, and 10th leaves from the base with each other. Ethylene production rates of individual leaves also declined over a 50-d period. However, as leaves began to appear chlorotic, a peak of ethylene production occurred that lasted for about 4 d followed by abscission. This peak involved a 3-fold or greater increase in the rate of ethylene production. The data indicate that intact leaves experience a climacteric-like surge in ethylene production after visible symptoms of senescence appear. This "ethylene climacteric" is apparently the signal that initiates hydrolysis of cell walls in the abscission zone.

Journal Article↗
PubMed1992

Enhanced Sensitivity to Ethylene in Nitrogen- or Phosphate-Starved Roots of Zea mays L. during Aerenchyma Formation.

Adventitious roots of maize (Zea mays L. cv TX 5855), grown in a well-oxygenated nutrient solution, were induced to form cortical gas spaces (aerenchyma) by temporarily omitting nitrate and ammonium (-N), or phosphate (-P), from the solution. Previously this response was shown (MC Drew, CJ He, PW Morgan [1989] Plant Physiology 91: 266-271) to be associated with a slower rate of ethylene biosynthesis, contrasting with the induction of aerenchyma by hypoxia during which ethylene production is strongly stimulated. In the present paper, we show that aerenchyma formation induced by nutrient starvation was blocked, under noninjurious conditions, by addition of low concentrations of Ag(+), an inhibitor of ethylene action, or of aminoethoxyvinyl glycine, an inhibitor of ethylene biosynthesis. When extending roots were exposed to low concentrations of ethylene in air sparged through the nutrient solution, N or P starvation enhanced the sensitivity to exogenous ethylene at concentrations as low as 0.05 microliters ethylene per liter air, promoting a more rapid and extensive formation of aerenchyma than in unstarved roots. We conclude that temporary deprivation of N or P enhances the sensitivity of ethylene-responsive cells of the root cortex, leading to cell lysis and aerenchyma.

Journal Article↗
PubMed1991

Growth factor-like effect of urokinase type plasminogen activator in human renal cells.

Human renal glomerular epithelial cells possess membrane urokinase receptors. Addition of purified active urokinase to these cells in serum free minimum medium induced a dose-dependent increase in 3H-thymidine incorporation and a doubling of cell number after 48 hours of incubation. Both receptor occupancy and enzymatic activity of u-PA were required to stimulate cell proliferation. This effect was inhibited by down regulation of protein kinase C (PKC) or by H7, an inhibitor of PKC. It involved a pertussis toxin-sensitive pathway. This effect of urokinase was additive with EGF but not with thrombin growth factor activity and was not inhibited by aprotinin, an inhibitor of plasmin.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Thrombin increases proliferation and decreases fibrinolytic activity of kidney glomerular epithelial cells.

Human glomerular epithelial cells (GECs) in culture synthesize single-chain, urokinase-type plasminogen activator (SC-uPA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor 1 (PAI-1) and possess specific membrane-binding sites for u-PA. Using purified 125I-alpha thrombin, we demonstrate here the presence of two populations of specific binding sites for thrombin on GECs (1.Kd = 4.3 +/- 1.0 x 10(-10) M, 5.4 +/- 1.4 x 10(4) M sites per cell, 2. Kd = 1.6 +/- 0.5 x 10(-8) M, 7.9 +/- 1.8 x 10(5) sites per cell). Purified human alpha thrombin promoted the proliferation of GECs and induced a time- and dose-dependent increase of SC-uPA, t-PA, and PAI-1 antigens released by GECs. Thrombin-mediated increase in antigen was paralleled by an increase in the levels of corresponding u-PA and PAI-1 messenger RNA. In contrast, thrombin decreased u-PA activity in conditioned medium. This discrepancy between u-PA antigen and u-PA activity was explained by a limited proteolysis of SC-uPA by thrombin, leading to a two-chain form detected by immunoblotting and that could not be activated by plasmin. Thrombin also decreased the number of u-PA binding sites on GECs (p less than 0.05) without changing receptor affinity. Hirudin inhibited the binding and the cellular effects of thrombin, whereas thrombin inactivated by diisopropylfluorophosphate had no effect, indicating that both membrane binding and catalytic activity of thrombin were required. We conclude that thrombin, through specific membrane receptors, stimulates proliferation of GECs and decreases the fibrinolytic activity of GECs both at the cell surface and in the conditioned medium. These results suggest that thrombin could be involved in the pathogenesis of extracapillary proliferation and persistency of fibrin deposits in crescentic glomerulonephritis.

Cell Division↗
PubMed1990

Thrombin regulates components of the fibrinolytic system in human mesangial cells.

Besides its procoagulant activity, thrombin has been shown to stimulate cell proliferation and to regulate the fibrinolytic pathway. We report here the effect of purified human alpha thrombin on the synthesis of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) by cultured human mesangial cells. Thrombin (0 to 2.5 U/ml) increased in a time- and dose-dependent manner the production of t-PA and PAI-1 (2- to 3-fold increase of secreted t-PA and PAI-1 release during a 24 hour incubation). This effect was associated with a twofold increase in DNA synthesis measured by 3H-thymidine incorporation. Zymographic analysis and reverse fibrin autography showed that thrombin also increased the level of the 110 Kd t-PA-PAI-1 complex, whereas PAI-1 was present as a free 50 Kd form in the culture medium conditioned by unstimulated and thrombin-stimulated cells. Free t-PA was never observed. Both membrane binding and catalytic activity of thrombin were required since the effects of 1 U/ml thrombin were inhibited by addition 2 U/ml hirudin, which inhibits the membrane binding and catalytic activity of thrombin, and since DFP-inactivated thrombin, which has the ability to bind but which has no enzymatic activity, did not induce t-PA or PAI-1. Gamma thrombin, which does not bind to thrombin receptor, did not increase t-PA and PAI-1 releases. The effects of thrombin were probably mediated by protein kinase C activation since H7, an inhibitor of protein kinases, inhibited significantly thrombin effects on t-PA and PAI-1 production, and since addition of an activator of protein kinase A, 8-bromocyclic AMP (100 microM), induced a significant inhibition of the thrombin effect. The effects of thrombin were also suppressed by 1.25 micrograms/ml alpha amanitin, suggesting a requirement of de novo RNA synthesis. Northern blot analysis indicated that thrombin induced an increase in the mRNA levels of t-PA and of PAI-1. We conclude that thrombin increases DNA synthesis in human mesangial cells and enhances the synthesis of both t-PA and PAI-1. The latter is released in a large excess as compared to t-PA. Hence, thrombin may have a role in provoking a localized hypofibrinolytic state and may contribute to the persistence of glomerular fibrin deposits during proliferative glomerulonephritis.

Blotting, Northern↗
PubMed1990

Does water deficit stress promote ethylene synthesis by intact plants?

The effect of plant water deficit on ethylene production by intact plants was tested in three species, beans (Phaseolus vulgaris L.), cotton (Gossypium hirsutum L.) and miniature rose (Rosa hybrida L., cv Bluesette). Compressed air was passed through glass, plant-containing cuvettes, ethylene collected on chilled columns, and subsequently assayed by gas chromatography. The usual result was that low water potential did not promote ethylene production. When plants were subjected to cessation of irrigation, ethylene production decreased on a per plant or dry weight basis of calculation. No significant promotion of ethylene production above control levels was detected when water deficit-treated bean or cotton plants were rewatered. The one exception to this was for cotton subjected to a range of water deficits, plants subjected to deficits of -1.4 to -1.6 MPa exhibited a transient increase of ethylene production of 40 to 50% above control levels at 24 or 48 hours. Ethylene was collected from intact leaves while plants developed a water deficit stress of -2.9 megapascals after rewatering, and no significant promotion of ethylene production was detected. The shoots of fruited, flowering cotton plants produced less ethylene when subjected to cessation of irrigation. In contrast, the ability of bench drying of detached leaves to increase ethylene production several-fold was verified for both beans and cotton. The data indicate that detached leaves react differently to rapid drying than intact plants react to drying of the soil with regard to ethylene production. This result suggests the need for additional attention to ethylene as a complicating factor in experiments employing excised plant parts and the need to verify the relevance of shock stresses in model systems.

Journal Article↗
PubMed1989

Urokinase synthesis and binding by glomerular epithelial cells in culture.

Fibrin deposits are frequently observed in the course of proliferative extracapillary glomerulonephritis and could be related to a defective local fibrinolysis. We studied human glomerular epithelial cells in culture which were found to release mainly a urokinase-type plasminogen activator (u-PA) identified on zymography by its molecular weight (53 kD), its plasminogen activator activity, and its neutralization by specific polyclonal anti-u-PA IgG. Trace amounts of tissue-type plasminogen activator (t-PA) complexed to a plasminogen activator inhibitor type 1 (PAI-1) were identified with specific antibodies. Specific binding sites were found at the surface of glomerular epithelial cells (kD: 2.10(-9) M), partially occupied by secreted u-PA. The spontaneous u-PA activity of the culture medium conditioned by glomerular epithelial cells was very low, suggesting that u-PA was released in its inactive single chain proenzyme form (SC-u-PA). After activation of SC-u-PA by plasmin, u-PA activity of the culture medium was found to increase in a time- and dose-dependent manner when cells were incubated with phorbol myristic acetate (PMA). This effect was inhibited by H7, a protein kinase C inhibitor. Stimulation of u-PA synthesis by PMA was also observed in two different epithelial tubular cell lines. LLC-PK1 and MDCK cells. However, 8 bromo cyclic AMP which increased u-PA release by LLC-PK1 cells was found to inhibit u-PA release by PMA-stimulated glomerular epithelial cells and MDCK cells. By Northern blot analysis we found that PMA induced an increase of u-PA mRNA level in glomerular epithelial cells and that cyclic AMP had an opposite effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗
PubMed1989

Decreased Ethylene Biosynthesis, and Induction of Aerenchyma, by Nitrogen- or Phosphate-Starvation in Adventitious Roots of Zea mays L.

Plants of Zea mays L. cv TX5855 were grown in a complete, well oxygenated nutrient solution then subjected to nutrient starvation by omitting either nitrate and ammonium or phosphate from the solution. These treatments induced the formation of aerenchyma close to the apex of the adventitious roots that subsequently emerged from the base of the shoot, a response similar to that shown earlier to be induced by hypoxia. Compared with control plants supplied with all nutrients throughout, N- or P-starvation consistently depressed the rates of ethylene release by excised, 25 mm apical segments of adventitious roots. Some enzymes and substrates of the ethylene biosynthetic pathway were examined. The content of 1-amino cyclopropane-1-carboxylic acid (ACC) paralleled the differences in ethylene production rates, being depressed by N or P deficiency, while malonyl-ACC showed a similar trend. Activity of ACC synthase and of ethylene forming enzyme (g(-1) fresh weight) was also greater in control roots than in nutrient starved ones. These results indicate that much of the ethylene biosynthetic pathway is slowed under conditions of N- or P-starvation. Thus, by contrast to the effects of hypoxia, the induction of aerenchyma in roots of Zea mays by nutrient starvation is not related to an enhanced biosynthesis and/or accumulation of ethylene in the root tips.

Journal Article↗