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Cytokinetic effects of estrogens in the human neuroblastoma cell line SK-ER3
THE STABILrrY OF THE COMPLEXES OF CITOSOL aH-ESTRADIOL A N D CITOSOL 3H-HYDROCORTISOL RELATED TO TEMPERATURE AND CONCENTRATION CHANGE~ OF KCI
Long-term effects of nifedypine in patients with essential hypertension and different patterns of cardiac geometry and hemodynamics. T. Zdrojewsk[, A. Kubasik, M. Dudziak, M. Stoniewska, B. Krupa~'~ciechowska. Medical University of Gdafisk, Gdafisk, Poland.
M. Abramovid1, Zofica Man,drz Inst#ut of Medical Chemistry z, Institute of Pharmacology and Toxicology, Faculty of Medicine 2, University of Nts, Brade Tasl~ovida 81, Ni~ Yugoslavia.
Our aim was to assess chronic effects of nifedypine on left ventricular (LV) morphology and function in patients with essential hypertension and different patterns of cardiac geometry. Baseline M-mode and two-dimensional echocardiography were obtained in seven patients with concentric remodeling (CR~ LV mass (LVM) normal; relative wall thickness (RWT) >2 SD above normal mean) and in ten patients with excentric nondilated hypertrophy (EH: LVM >2SD above normal mean; RWT normal) before and one year after therapy with nifedypine. Before therapy systemic hemodynamics paralleled LV geometry changes: total peripheral resistance index (TPRI, mmHg/min/r 1 per m2) was higher, and cardiac index (CI, ml/min per m2) and end-systolic stress (ESS, g/cm 2) were lower in patients with CR when compared to patients with EH. Therapy with nifedypine normalized RWT, CI, TPRI and ESS in patients Cwith _ R and regressed LVM (g) and TPRI in patients with EH._
The stability of complexes of 3H-estradiol and 3H-hydrocortisol, with receptor components of liver and rat uterus cells was examined by testing their resistance against increased concentration of salts and temperature changes. For experiments we used female rats (Mill Hill Hooded) 2-3 months old, about 250g body weight. As biological materials the samples of tissue of liver and uterus were used 10 ml soluble cytoplsmatic fraction from liver and uterus was incubated at 20°C and 37°C for 30 min with 20[,tCi 3H-estradiol or 20[,tCi-hydrocortisoL The nespecfficaly bond hormones were eliminated by double pretreatment with dextra-Carcoal. The stability of formed complexes of estradiol and hydrocortisol against increased concentrations of KCI was examined by addition of KCI up to physiological concentrations of: 0.1; 0.3; 0.5; 1; 3 and 5 tool/l; after that the samples were dialyzed in TE-solution (1:200) for 18-20h, The stability of complexes was determined from the ratio of dissociated moleculs and the radioactivity of bonded steroids. The temperature stability of created complexes at 20°C and 37°C was tested by abrupt changes of temperature: some samples were frozen and others warmed 15 rain at 40°C. After that samples were dialyzed 20h at 4°C. The radioactivity of bonded hormones was determined in all examined samples by using Bray's solution. The measurement was performed on Beckman LS-100 counter. The complexes of both steroid hormones formed at 20°C were more stable than complexes formed at 37°C regarding both: KCI concentration changes and abrupt changes of temperature.
CR before CR after ly EH before EH after ly__ MAP 109+~ 99-+3* 117-+3# 104+9' LVM 210_--h6 197-+19 278_+9# 238-+17" CI 1564-+71 3327_+308" 2417-+154# 2787+_217 TPRI 70+_3 37+_5* 50+_3# 39+_3* ESS 25+_3 495:10" 43+_3# 43+--5 mean _+SEM; * p<0.05 vs. respective group before therapy; # p<0.05 vs. CR before therapy; MAP -mean arterial pressure, mmHg
Thus long-term therapy with nifedipine resulted in normalization of LV mass, geometry, hemodynamics and ventricular load in respect to pretreatment abnormalities.
FLOW CYTOMETRIC DETECTION OF LIPOCORTIN 1 B I N D I N G SITES ON RAT ANTERIOR PITUITARY CELLS. H.C.Chfistian. 1N.I.Goulding, 2R.J.Flower & J.C.Buckingham. Department of Pharmacology, Chafing Cross & Westminster Medical School, London W6 8RF and Departments of Rheumatology & 2Biochemical Pharmacology, St Bartholomew's Hospital Medical College, London ECIM 6BQ.
CYTOKINETIC EFFECTS OF ESTROGENS IN THE HUMAN NEUROBLASTOMA CELL LINE SK-ER3 P. Agrati, M.G. Bottone°, Z.Q Ma, C. Patrone, C. Pellicciari° and A. Maggi Institute of Pharmacological Sciences, Via Balzaretti 9, 20133 Milano and °Dip. Animal Biology, Piazza Botta 10, 27100 Pavia, Italy
Lipocortin 1 (LC1) is an important mediator of glucocorticoid action in the rat anterior pituitary gland (l), but its molecular mode o f action is obscure. The present study aimed to determine whether LC1 binding sites analogous to those charactefised on the surface of human peripheral blood PMNs and monocytes (2) are expressed on anterior pituitary cells, Aliquots of rat anterior pituitary cells (obtained post mortem and dispersed enzymatically with 0.l% collagenase) were incubated for lh at 4°C in 96 well plates (0.5 - 1.0 million cells/well) with human recombinant LC1 (0.001-0.5~aM). LC1 molecules bound to the cell surface were detected by sequential incubation with a specific monoclonal anti-LC1 antibody (anti-LC1 mAb, coded 1B) and a fluorescent labelled (FITC) second antibody (anti-mouse IgG Ab) and quantified by computerised fluorescence activated flow cytometry. Concentration-dependent (0.5nM-0.5gM), saturable LC1 binding was observed with an estimate Kd of 13.65 + 1.8nM (n=4). At saturation (0.5gM LC1), the total number of binding sites/cell was 140,800 -+ 14,960 (mean + SE, n=8) with a non-specific component (mAb 1B alone) of 3,650 + 745 (n=8). Binding was Ca++ and temperature dependent and destroyed by pre-incubation (30 rain) of the cells with trypsin (0.025%). The results demonstrate for the first time the presence of cell surface LC1 binding sites in the anterior pituitary gland. Further studies are now required to investigate the LC1 binding characteristics of the various pituitary cell populations.
To investigate the role of estrogens in cells of nervous origin, the neuroblastoma cell line SK-N-BE was stably transfected for the estrogen receptor, generating a cell line named SK-ER3. After 1713estradiol administration, the proliferation of SK-ER3 cells was blocked, while morphological and biochemical evidence of differentiation was found 1 To further to analyze the cytokinetic effect of estrogen treatments on SK-ER3 cells, we undertook a dualparameter flow cytometric study of DNA content versus bromodeoxyuridine (BrdU) incorporation. Control and estrogentreated cells were pulse labelled with 40~tM BrdU,for 30 min., then harvested by mild trypsinization and fixed with cold 70% ethanol. Ceils were indirectly FITC-hnmtmolabelled with an anti-BrdU antibody and counterstained for DNA with 5 ug/ml propidium iodide. Dual-parameter cytometric determinations showed that, after estrogen tratment, significantly fewer cells entered S-phase, while cells accumulated in GO/I phase of the cells cycle. This is consistent with previous observations o n 3H-thymidine incorporation 1. The irnmunocytochemical detection of cycle related protein (e.g. Ki-67 and PCNA) will provide additional informations on the possible exit of ceils from the cycling compartment into a GO quiescent state. Ackn~owledgements The present work was supported by P.F. Biotechilologies and Bioinstrumentation (AM) and Italian Association Cancer Research (AIRC).
This work was generously supported by the Wellcome (041943/Z/94/MP/JF) and the Isle of Man Department of Education 1. 2.
1. Ma Z.Q. et al. Proc. Natl. Acad. Sci. (USA)90:3740-3744, 1993
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Trust
Taylor, A.D., Cowell, A-M,, Flower, R.J. & Buckingham J.C. (1993). Neuroendocrinology 58,430-439. Perretti, M., Flower, R.J. & Goulding, N.J. (1993). Biochem. Biophys. Res Comm., 192, 345-350.
Long-term effects of nifedypine in patients with essential hypertension and different patterns of cardiac geometry and hemodynamics. T. Zdrojewsk[, A. Kubasik, M. Dudziak, M. Stoniewska, B. Krupa~'~ciechowska. Medical University of Gdafisk, Gdafisk, Poland.
M. Abramovid1, Zofica Man,drz Inst#ut of Medical Chemistry z, Institute of Pharmacology and Toxicology, Faculty of Medicine 2, University of Nts, Brade Tasl~ovida 81, Ni~ Yugoslavia.
Our aim was to assess chronic effects of nifedypine on left ventricular (LV) morphology and function in patients with essential hypertension and different patterns of cardiac geometry. Baseline M-mode and two-dimensional echocardiography were obtained in seven patients with concentric remodeling (CR~ LV mass (LVM) normal; relative wall thickness (RWT) >2 SD above normal mean) and in ten patients with excentric nondilated hypertrophy (EH: LVM >2SD above normal mean; RWT normal) before and one year after therapy with nifedypine. Before therapy systemic hemodynamics paralleled LV geometry changes: total peripheral resistance index (TPRI, mmHg/min/r 1 per m2) was higher, and cardiac index (CI, ml/min per m2) and end-systolic stress (ESS, g/cm 2) were lower in patients with CR when compared to patients with EH. Therapy with nifedypine normalized RWT, CI, TPRI and ESS in patients Cwith _ R and regressed LVM (g) and TPRI in patients with EH._
The stability of complexes of 3H-estradiol and 3H-hydrocortisol, with receptor components of liver and rat uterus cells was examined by testing their resistance against increased concentration of salts and temperature changes. For experiments we used female rats (Mill Hill Hooded) 2-3 months old, about 250g body weight. As biological materials the samples of tissue of liver and uterus were used 10 ml soluble cytoplsmatic fraction from liver and uterus was incubated at 20°C and 37°C for 30 min with 20[,tCi 3H-estradiol or 20[,tCi-hydrocortisoL The nespecfficaly bond hormones were eliminated by double pretreatment with dextra-Carcoal. The stability of formed complexes of estradiol and hydrocortisol against increased concentrations of KCI was examined by addition of KCI up to physiological concentrations of: 0.1; 0.3; 0.5; 1; 3 and 5 tool/l; after that the samples were dialyzed in TE-solution (1:200) for 18-20h, The stability of complexes was determined from the ratio of dissociated moleculs and the radioactivity of bonded steroids. The temperature stability of created complexes at 20°C and 37°C was tested by abrupt changes of temperature: some samples were frozen and others warmed 15 rain at 40°C. After that samples were dialyzed 20h at 4°C. The radioactivity of bonded hormones was determined in all examined samples by using Bray's solution. The measurement was performed on Beckman LS-100 counter. The complexes of both steroid hormones formed at 20°C were more stable than complexes formed at 37°C regarding both: KCI concentration changes and abrupt changes of temperature.
CR before CR after ly EH before EH after ly__ MAP 109+~ 99-+3* 117-+3# 104+9' LVM 210_--h6 197-+19 278_+9# 238-+17" CI 1564-+71 3327_+308" 2417-+154# 2787+_217 TPRI 70+_3 37+_5* 50+_3# 39+_3* ESS 25+_3 495:10" 43+_3# 43+--5 mean _+SEM; * p<0.05 vs. respective group before therapy; # p<0.05 vs. CR before therapy; MAP -mean arterial pressure, mmHg
Thus long-term therapy with nifedipine resulted in normalization of LV mass, geometry, hemodynamics and ventricular load in respect to pretreatment abnormalities.
FLOW CYTOMETRIC DETECTION OF LIPOCORTIN 1 B I N D I N G SITES ON RAT ANTERIOR PITUITARY CELLS. H.C.Chfistian. 1N.I.Goulding, 2R.J.Flower & J.C.Buckingham. Department of Pharmacology, Chafing Cross & Westminster Medical School, London W6 8RF and Departments of Rheumatology & 2Biochemical Pharmacology, St Bartholomew's Hospital Medical College, London ECIM 6BQ.
CYTOKINETIC EFFECTS OF ESTROGENS IN THE HUMAN NEUROBLASTOMA CELL LINE SK-ER3 P. Agrati, M.G. Bottone°, Z.Q Ma, C. Patrone, C. Pellicciari° and A. Maggi Institute of Pharmacological Sciences, Via Balzaretti 9, 20133 Milano and °Dip. Animal Biology, Piazza Botta 10, 27100 Pavia, Italy
Lipocortin 1 (LC1) is an important mediator of glucocorticoid action in the rat anterior pituitary gland (l), but its molecular mode o f action is obscure. The present study aimed to determine whether LC1 binding sites analogous to those charactefised on the surface of human peripheral blood PMNs and monocytes (2) are expressed on anterior pituitary cells, Aliquots of rat anterior pituitary cells (obtained post mortem and dispersed enzymatically with 0.l% collagenase) were incubated for lh at 4°C in 96 well plates (0.5 - 1.0 million cells/well) with human recombinant LC1 (0.001-0.5~aM). LC1 molecules bound to the cell surface were detected by sequential incubation with a specific monoclonal anti-LC1 antibody (anti-LC1 mAb, coded 1B) and a fluorescent labelled (FITC) second antibody (anti-mouse IgG Ab) and quantified by computerised fluorescence activated flow cytometry. Concentration-dependent (0.5nM-0.5gM), saturable LC1 binding was observed with an estimate Kd of 13.65 + 1.8nM (n=4). At saturation (0.5gM LC1), the total number of binding sites/cell was 140,800 -+ 14,960 (mean + SE, n=8) with a non-specific component (mAb 1B alone) of 3,650 + 745 (n=8). Binding was Ca++ and temperature dependent and destroyed by pre-incubation (30 rain) of the cells with trypsin (0.025%). The results demonstrate for the first time the presence of cell surface LC1 binding sites in the anterior pituitary gland. Further studies are now required to investigate the LC1 binding characteristics of the various pituitary cell populations.
To investigate the role of estrogens in cells of nervous origin, the neuroblastoma cell line SK-N-BE was stably transfected for the estrogen receptor, generating a cell line named SK-ER3. After 1713estradiol administration, the proliferation of SK-ER3 cells was blocked, while morphological and biochemical evidence of differentiation was found 1 To further to analyze the cytokinetic effect of estrogen treatments on SK-ER3 cells, we undertook a dualparameter flow cytometric study of DNA content versus bromodeoxyuridine (BrdU) incorporation. Control and estrogentreated cells were pulse labelled with 40~tM BrdU,for 30 min., then harvested by mild trypsinization and fixed with cold 70% ethanol. Ceils were indirectly FITC-hnmtmolabelled with an anti-BrdU antibody and counterstained for DNA with 5 ug/ml propidium iodide. Dual-parameter cytometric determinations showed that, after estrogen tratment, significantly fewer cells entered S-phase, while cells accumulated in GO/I phase of the cells cycle. This is consistent with previous observations o n 3H-thymidine incorporation 1. The irnmunocytochemical detection of cycle related protein (e.g. Ki-67 and PCNA) will provide additional informations on the possible exit of ceils from the cycling compartment into a GO quiescent state. Ackn~owledgements The present work was supported by P.F. Biotechilologies and Bioinstrumentation (AM) and Italian Association Cancer Research (AIRC).
This work was generously supported by the Wellcome (041943/Z/94/MP/JF) and the Isle of Man Department of Education 1. 2.
1. Ma Z.Q. et al. Proc. Natl. Acad. Sci. (USA)90:3740-3744, 1993
--81
--
Trust
Taylor, A.D., Cowell, A-M,, Flower, R.J. & Buckingham J.C. (1993). Neuroendocrinology 58,430-439. Perretti, M., Flower, R.J. & Goulding, N.J. (1993). Biochem. Biophys. Res Comm., 192, 345-350.