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Biomedical subjects

G S Whitley

Publications and source records attributed to G S Whitley.

At least 37 records · Page 2Linked to original sources
PubMed1994

Effect of anti-fungal imidazoles on mRNA levels and enzyme activity of inducible nitric oxide synthase.

1. Experiments were performed to examine the effects of anti-fungal imidazole compounds (clotrimazole, econazole and miconazole) on the induction of nitric oxide (NO) synthase and subsequent production of NO in the cultured murine monocyte/macrophage cell line J774 using a specific cDNA probe for inducible NO synthase mRNA and by monitoring nitrite production. 2. Stimulation of J774 cells with lipopolysaccharide (LPS, 10 micrograms ml-1) resulted in the induction of NO synthase activity as determined by nitrite accumulation in the culture medium (48 +/- 3 nmol per 10(6) cells over 24 h). Production of nitrite was inhibited by co-incubation of cells with LPS (10 micrograms ml-1) and either dexamethasone (10 microM) or NG-monomethyl-L-arginine (L-NMMA; 0.1 mM), however, only L-NMMA was an effective inhibitor of nitrite production when added after induction of NO synthase had occurred. 3. Co-incubation of J774 cells with LPS (10 micrograms ml-1) and either clotrimazole, econazole or miconazole (1-10 microM) resulted in a concentration-dependent inhibition of nitrite production over the subsequent 24 h without any evidence for a cytotoxic effect. However, addition of these imidazoles after induction of NO synthase did not inhibit nitrite production. 4. Messenger RNA for inducible NO synthase was not detected in unstimulated J774 cells. Treatment with LPS (10 micrograms ml-1) for 4 h resulted in significant expression of mRNA for inducible NO synthase which was not altered in the presence of econazole (10 microM) but was reduced significantly by dexamethasone (10 microM). 5. These results demonstrate that anti-fungal imidazoles inhibit the production of nitric oxide by cultured J774 cells by a mechanism which appears to differ from that of dexamethasone and substrate type inhibitors of NO synthase. Furthermore, the presence of mRNA for NO synthase does not indicate the presence of functionally active NO synthase.

Amino Acid Oxidoreductases↗
PubMed1993

Nitric oxide stimulates cyclic GMP in human thyrocytes.

Sodium nitroprusside spontaneously breaks down in solution to produce the vasodilator nitric oxide. In many cell types, this stimulates the cytosolic form of the enzyme guanylate cyclase, resulting in the elevation of cyclic GMP (cGMP). We have investigated the effect of sodium nitroprusside on the generation of cGMP in primary human thyrocytes and the SV40-transfected human thyroid cell line SGHTL-189. A dose-dependent increase in cGMP was obtained and the maximum response was observed with concentrations above 10 microM sodium nitroprusside in both cell types. Methylene blue (50 microM) had no significant effect on basal cGMP production but inhibited the effect of sodium nitroprusside at all concentrations tested, thus demonstrating that the effect was due to nitric oxide. Sodium nitroprusside had no effect on cyclic AMP (cAMP) production in these cells. TSH at 100 and 1000 microU/ml significantly stimulated the production of cAMP, but not that of cGMP, in primary human thyrocytes. Sodium nitroprusside had no significant effect on basal or TSH-stimulated triiodothyronine secretion in primary human thyrocytes. Forskolin (10 microM) significantly stimulated cAMP production in both primary thyrocytes and SGHTL-189 cells. Although forskolin had no significant effect on basal cGMP production, sodium nitroprusside-stimulated cGMP production was significantly reduced by forskolin. However, this inhibitory effect was not related to the production of cAMP.

Cells, Cultured↗
PubMed1993

The effect of basic fibroblast growth factor on the growth and function of human thyrocytes.

Basic fibroblast growth factor (bFGF) was quantitated in human primary thyrocyte cultures and thyroid cell lines produced by transfection with pSV3neo. Immunoreactive-bFGF (ir-bFGF) bound to heparin-Sepharose affinity columns eluted with 1.8-2.0 mol NaCl/l and had a molecular weight of approximately 17,000. Recombinant human bFGF in the presence of 5% serum increased the growth of transfected human thyrocytes but not the growth of primary human thyrocytes. Preincubation of cells with up to 100 micrograms bFGF/l potentiated TSH-stimulated cAMP release from the transfected cells but inhibited release from primary human thyroid cultures. bFGF may be an important modulator of thyroid cell function and growth.

Cells, Cultured↗
PubMed1992

Characterization of human umbilical vein endothelial cell lines produced by transfection with the early region of SV40.

Human umbilical vein endothelial cells were transfected by electroporation with the plasmid pSV3neo, containing the early region of simian virus 40. The resultant "cell lines" divide rapidly (population doubling time of 33 h) for up to 24 passages in medium supplemented with 5% (v/v) serum and 2.5 micrograms/ml endothelial cell growth supplement. Several of these lines express basal levels of ICAM-1 and MHC class I but not MHC class II. One cell line, designated SGHEC-7, retained a number of differentiated endothelial cell functions throughout its lifespan. These functions include increased production of tissue plasminogen activator in response to histamine, thrombin, and PMA. Stability of function and rapid growth over 24 passages endow these cells with a number of advantages over primary cultures. The homogeneous cell population and consistency of response make them ideal for biochemical and immunological studies hereto impractical with primary human endothelial cells. The success of this approach may allow the production of functional cell lines from other vascular beds.

Cell Adhesion Molecules↗
PubMed1991

Effects of ageing on the growth and differentiated function of transfected human thyrocytes.

Senescence in primary cultures of mammalian cells is characterised by cessation of growth after a number of cell divisions; this may be associated with loss of some differentiated functions. Recent studies on bovine adrenocortical cells have suggested that expression of simian virus-40 (SV40) early region in these cells may prevent phenotypic losses due to senescence. We report here data on growth and differentiated function of two human thyrocyte cell lines (SGHTL-34 and -45) generated by the transfection of primary thyrocytes with the plasmid pSV3neo which contains the SV40 early region. Growth was assessed by fluorometric DNA estimations and calculation of cell population doubling time; function was assessed by binding studies using 125I-bovine thyrotrophin (TSH) and measurement of cyclic adenosine 3',5'-monophosphate (cAMP) response to stimulation with TSH, forskolin and cholera toxin. After 3-12 months in stable culture there was a gradual increase in the doubling time of both cell lines over a 3-month period (SGHTL-34 cells, early 34.5 +/- 4.5 h, late 301 +/- 111.6 h; SGHTL-45 cells, early 53.4 +/- 4.4 h, late 148.3 +/- 26.3 h; mean +/- SEM). Scatchard analysis demonstrated a loss of the high affinity TSH receptor over the same time period. The increase in cAMP in response to 1000 microU/ml TSH declined until the cells became unresponsive (SGHTL-34 early, cAMP 10.3 +/- 0.7 pmol/well; late, cAMP -0.4 +/- 0.3 pmol/well; SGHTL-45 early, cAMP 11.3 +/- 1.1 pmol/well, late, cAMP 0.3 +/- 0.1 pmol/well). The cAMP responses to forskolin and cholera toxin were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1990

Expression of intercellular adhesion molecule-1 (ICAM-1) on human thyroid cells lines correlated with their binding of lymphoblasts.

We have investigated the expression of intercellular adhesion molecule-1 (ICAM-1) by novel functional human thyroid cell lines (designated SGHTL). ICAM-1 is constitutively expressed and it is rapidly upregulated in response to each of the recombinant cytokines: gamma-interferon, interleukin-1 and tumour necrosis factor. This contrasts with the more slowly increased expression of major histocompatibility complex (MHC) class II antigens in response to gamma-interferon alone. We have also demonstrated binding of activated lymphocytes to SGHTL cells: this interaction is increased following treatment with these cytokines and is inhibited by monoclonal antibodies directed against ICAM-1 or lymphocyte function-associated antigen-1 (LFA-1) but not by antibodies against CD2 or MHC class II antigens. Hence, we conclude that the binding of lymphoblasts to human thyroid cells involves an LFA-1- and ICAM-1-dependent pathway as well as other basal and cytokine-inducible pathway(s). These do not appear to involve MHC class II antigens, CD2 or an LFA-1 ligand other than ICAM-1.

Antibodies, Monoclonal↗
PubMed1990

Modulation of the biological activity of thyrotrophin by anti-human thyrotrophin monoclonal antibodies.

The mechanism by which monoclonal antibodies enhance the biological activity of a number of hormones is poorly understood. One such antibody (GC73), which binds to human but not bovine TSH, enhances the bioactivity of human TSH in vivo. We have investigated whether GC73 enhancement of TSH bioactivity involves potentiation of hormone-receptor activation assessed by the cyclic AMP (cAMP) responses of both primary human thyrocyte cultures and a TSH-responsive human thyrocyte cell line (SGHTL-45). GC73 had no effect on basal cAMP production. In contrast to its enhancement of the bioactivity of human TSH in vivo, it markedly inhibited the cAMP response to 1 and 10 mU human TSH/ml in primary thyrocytes. This effect was dose-dependent with neutralization of the bioactivity of TSH occurring at 2 mg GC73/ml. GC73 had no effect on the bioactivity of bovine TSH. In contrast, a second anti-TSH monoclonal antibody (TC12), which binds to both human and bovine TSH, inhibited the bioactivity of both species of TSH. Similar results were obtained using SGHTL-45 cells, although the peak concentrations of cAMP were lower. We conclude that binding of GC73 to human TSH resulted in inhibition rather than enhancement of the in-vitro biological activity of human TSH. We suggest that GC73 enhancement of human TSH bioactivity seen in vivo does not result from a mechanism involving potentiation of receptor activation by human TSH.

Antibodies, Monoclonal↗
PubMed1990

Binding of 125I-labelled thyrotrophin to transfected human thyrocytes (SGHTL-34 cells).

The binding of 125I-labelled bovine TSH to a human thyroid cell line (SGHTL-34) has been studied. Binding to hormonally responsive cells was time dependent, specific and reversible. Scatchard analysis of the binding data indicated the presence of a single binding site with high affinity (intrinsic dissociation constant (Kd) = 0.25 +/- 0.08 nmol/l; mean +/- S.E.M.; n = 4) and low capacity (maximum binding (Bmax) = 104 +/- 29 fmol/mg protein; mean +/- S.E.M.; n = 4). Hill plots confirmed the presence of a single site. Kinetic data demonstrated close agreement between the Kd and Bmax obtained from the competition data (Kd = 0.23 +/- 0.35 nmol/l; Bmax = 161 +/- 83 fmol/mg protein; n = 6).

Cell Line↗
PubMed1989

Cytotoxic effects of amiodarone and desethylamiodarone on human thyrocytes.

Since recent in vivo evidence suggests that the benzofuran antiarrhythmic drug amiodarone has a direct toxic effect on the human thyroid gland, we have investigated the effects of both amiodarone and its metabolite desethylamiodarone on a novel immortalized functional human thyrocyte line (SGHTL-34 cells). Desethylamiodarone markedly reduced cell number as assessed from both DNA and protein content. Few cells were left after 24 hr exposure to 12.5 micrograms/ml; the concentration producing death of 50% of cells (EC50) was 6.8 +/- 1.1 micrograms/ml (mean +/- SE, N = 15). Amiodarone was much less potent, producing a maximum decrease in cell number of approximately 25% at concentrations up to 50 micrograms/ml. The effect of desethylamiodarone was seen within 24 hr of culture. T3 in concentrations up to 0.75 micrograms/ml had no effect on the action of amiodarone or desethylamiodarone on SGHTL-34 cells. Light microscopy demonstrated vacuolation of SGHTL-34 cells after 4-day culture with either the drug or its metabolite. Studies using primary cultures of human retroorbital fibroblasts demonstrated that the greater cytotoxicity of desethylamiodarone was not confined to thyrocytes. When SGHTL-34 cells were incubated with 2.5 micrograms/ml desethylamiodarone for 4 days, 71.7 +/- 0.9% was taken up by the cells; there was no detectable conversion to amiodarone. Incubation of thyrocytes with 50 micrograms/ml amiodarone for 4 days resulted in the uptake of 80.1 +/- 2.1% by the cells. In addition, 5.0 +/- 0.1% of the amiodarone was converted to material with the same retention time as desethylamiodarone standard; of this material, 72.9 +/- 2.8% was taken up by the cells. We conclude that desethylamiodarone, at concentrations near those found in the plasma of patients on long-term amiodarone therapy, exerts a direct cytotoxic effect on human thyroid cells in short-term culture. This effect may play a role in the aetiology of clinical thyroid disease during amiodarone therapy. We suggest that, since the effect is not restricted to thyrocytes, desethylamiodarone may play a role in the aetiology of amiodarone toxicity which occurs clinically in many tissues.

Amiodarone↗
PubMed1989

Immune induction of major histocompatibility complex antigens on a human thyroid cell line (SGHTL-34).

Expression of major histocompatibility complex antigens by epithelial cells may play a role in the aetiology of autoimmune disorders. We have studied the effect of gamma-interferon on SGHTL-34, a human thyroid cell line which constitutively expresses class I but not class II antigens. gamma-Interferon induced the expression of class II and increased the expression of class I molecules (assessed by flow cytofluorimetry) in a dose-dependent manner. Thyrotrophin or phytohaemagglutinin had no effect on either class I or class II expression. However, a supernatant from phytohaemagglutinin-stimulated peripheral blood mononuclear cells, containing 6400 U gamma-interferon/ml, was an effective inducer of both class I and class II antigens. These data clarify earlier studies using primary thyroid cultures, which are contaminated with cells of the immune system.

Cell Line↗
PubMed1989

The interaction of arginine vasopressin and oxytocin with bovine adrenal medulla cells.

Binding of [3H]arginine vasopressin (AVP) and [3H]oxytocin to primary monolayer cultures of bovine adrenal chromaffin cells was time-dependent, and the binding sites for each peptide were specific and saturable. Studies with the V1 AVP antagonist d(CH2)5Tyr(Me)2-AVP, the V2 agonist 1-deamino-8-D-AVP and the V2 antagonist d(CH2)5D-Leu2,Val4-AVP indicated that the AVP receptor was V1 in specificity. Scatchard plots showed that each ligand interacted with a single high-affinity, low-capacity binding site: oxytocin dissociation constant (Kd) 0.29 +/- 0.02 nmol/l, maximum binding capacity (Bmax) 7.6 +/- 0.2 fmol/10(6) cells (or 4500 +/- 102 sites/cell) (n = 3); AVP Kd 0.09 +/- 0.02 nmol/l, Bmax 5.1 +/- 0.63 fmol/10(6) cells (or 3050 +/- 318 sites/cell) (n = 3). Although forskolin in concentrations from 1 nmol/l to 1 mmol/l stimulated cyclic AMP (cAMP) production in isolated chromaffin cells, this did not result in detectable catecholamine release. Neither AVP nor oxytocin in concentrations between 10 pmol/l and 10 mumols/l stimulated cAMP production in these cells. Vasopressin in concentrations as low as 10 pmol/l stimulated a sixfold increase in total inositol phosphates; the dose-response curve was triphasic. Oxytocin had little effect on total inositol phosphate accumulation at low concentrations, but concentrations above micromolar stimulated total inositol phosphate production approximately fourfold. There was no measurable release of catecholamines in response to either peptide. The physiological consequences of these AVP-induced changes in inositol phosphate concentrations remain to be elucidated.

Adenylyl Cyclases↗
PubMed1989

Differential role for ADP-ribosylation in gene expression during the activation of T lymphocytes by various stimuli.

Simple competitive inhibitors of the nuclear enzyme ADP-ribosyltransferase, such as 3-methoxybenzamide (3MB), are known to block mitogen-induced activation of lymphocytes by inhibiting an early event. We now report that 3MB affects neither the generation of inositol phosphates nor the increase in cytoplasmic calcium in human T lymphocytes stimulated with phytohaemagglutinin (PHA), indicating that it acts on later or parallel events. The proliferative response to the phorbol ester 12-o-tetradecanoylphorbol-13-acetate (TPA) was much less sensitive to 3MB than was the response to PHA. Similarly, the TPA-induced increases in the expression of the c-myc proto-oncogene and of the Tac polypeptide of the interleukin-2 receptor were not affected by 3MB, whereas the same responses to either PHA or the calcium ionophore A23187 were inhibited by 3MB. The data suggest that 3MB affects the calcium-mediated signal for T-lymphocyte activation, acting after the increase in cytoplasmic calcium, and possibly also affects other signal transduction pathways distinct from the hydrolysis of polyphosphoinositides. There are some similarities between the actions of 3MB and cyclosporin.

Adenosine Diphosphate Ribose↗
PubMed1988

An analogue of creatine increases TRH-stimulated prolactin secretion and phosphoinositide hydrolysis in rat pituitary tumor cells.

Prolactin (PRL)-secreting GH3 cells were grown, in vitro, with the creatine analogue beta-guanidinopropionic acid (GPA) added to the culture medium. After 5 days there was a small increase in basal and greatly increased thyrotropin-releasing hormone (TRH)-stimulated PRL secretion. The site of action of GPA is at the TRH-induced hydrolysis of phosphoinositides, since increased amounts of mono, bis and tris/tetrakis inositol phosphates were found in treated cells, while the PRL secretion induced by a phorbol ester or a calcium ionophore, treatments which mimic the second messages generated by inositol phospholipid hydrolysis, were not enhanced by GPA. The mechanism by which GPA increases phospholipase C activity has not been fully elucidated but may involve the activity of a controlling G protein.

Aminoquinolines↗
PubMed1987

SGHTL-34, a thyrotrophin-responsive immortalised human thyroid cell line generated by transfection.

Normal human thyrocytes were immortalised by electroporation-mediated transfection with the plasmid pSV3neo. One resultant cell line (SGHTL-34) contains approximately 10 copies of simian virus 40 (SV40) early region incorporated into the genome and has been in continuous monolayer culture for 9 months. SGHTL-34 cells grow rapidly (doubling time 30 h) and contain cytokeratin filaments. They demonstrate a morphological change in response to thyrotrophin (TSH) and possess a TSH- and forskolin-sensitive adenylate cyclase, the thresholds for stimulation being 10 microU/ml bovine TSH- and forskolin-sensitive development of this novel human thyrocyte cell line may allow further study of the regulation of human thyroid growth and differentiation.

Adenylyl Cyclases↗
PubMed1986

Effects of alpha-melanocyte-stimulating hormone on the cyclic AMP and phospholipid metabolism of rat adrenocortical cells.

Results on the effects of peptides on the phospholipid metabolism and steroid and cyclic AMP (cAMP) outputs of rat adrenal capsular cells (96% zona glomerulosa, 4% zona fasciculata) were obtained in a series of three batch experiments. Their significance was examined by analysis of variance. Incorporation of [32P] into phosphatidylcholine, phosphatidic acid and phosphatidylinositol was measured. Production of [3H]inositol-1 monophosphate, inositol-1,4 bisphosphate and inositol-1,4,5 tris-phosphate was estimated after prelabelling with [3H]inositol followed by 1 min incubation with a steroidogenic stimulus. Angiotensin II (0.25 nmol/l to 0.25 mumol/l) highly significantly (P less than 0.01) stimulated aldosterone and corticosterone outputs, [32P] incorporation into phosphatidic acid and phosphatidylinositol (but not into phosphatidylcholine) and the production of the three [3H]inositol phosphates. Aldosterone and corticosterone outputs were stimulated by alpha-MSH (above 0.1 nmol/l). However, incorporation of [32P] was not significantly increased until 10 mumol alpha-MSH/l but, unlike with angiotensin II, incorporation into phosphatidylcholine was also then stimulated. Also, the production of the inositol phosphates was not increased significantly (P greater than 0.05) by any dose of alpha-MSH (10 nmol/l, 1 mumol/l and 0.1 mmol/l) used. Therefore, it can be concluded that alpha-MSH does not stimulate phospholipase C in rat zona glomerulosa cells. In further experiments, it was also found that there were significant increases in cAMP as well as in steroid outputs above 1 nmol alpha MSH/l (highly significant above 10 nmol alpha-MSH/l). There were plateaux of the outputs of both steroids and cAMP from 0.1 to 1 mumol alpha-MSH/l. However, there were further increases in steroid and cAMP outputs of the capsular cells at higher doses. Concomitant results on the stimulation of corticosterone output by zona fasciculata-reticularis cells indicate that this additional increase was mostly due to the stimulation of the contaminating zona fasciculata cells. It was also confirmed that alpha-MSH preferentially stimulates steroidogenesis by the zona glomerulosa. However, under our conditions, alpha-MSH highly significantly increased the output of cAMP by both zona fasciculata and glomerulosa cells.

Adrenal Cortex↗