Altered maternal hypothalamic-pituitary-adrenal axis activity in obese pregnancy is associated with macrosomia and prolonged pregnancy.

BACKGROUND: The hypothalamic-pituitary-adrenal (HPA) axis is important for fetal growth and timing of parturition. Maternal obesity is associated with macrosomia (birthweight ⩾4000g) and prolonged pregnancy (⩾41weeks). We aimed to characterise HPA axis hormones in obese pregnancy and to test associations with these pregnancy outcomes. METHOD: Fasting cortisol was measured by radioimmunoassay in venous blood at 16, 28 and 36 weeks of gestation in 286 obese (BMI 44.05±3.98kg/m(2)) and 137 lean (BMI 22.71±1.66kg/m(2)) pregnant women. In subsets (n=20 obese, 20 lean) we measured corticosteroid binding globulin (CBG) and CRH by radioimmunoassay; progesterone, estradiol (E2), estriol (E3) and sex-hormone-binding-globulin (SHBG) by ELISA; and albumin by bromocresol green binding. Free cortisol levels were calculated using Coolen's equation. RESULTS: Cortisol, CBG, calculated free cortisol, CRH, E2, E3, progesterone and SHBG levels rose similarly during pregnancy in obese and lean, but were significantly lower in obese (p<0.05). In obese, lower free cortisol at 16 weeks was associated with higher birthweight (r=-0.46, p<0.05). Cortisol was not associated with labour onset. CRH was significantly lower at 36 weeks in women who delivered at ⩾41weeks and in women with macrosomic babies (p<0.05); and correlated negatively with gestation at delivery in obese (r=-0.557, p<0.05). CONCLUSION: Our findings suggest that decreased HPA axis activity in obese pregnancy may be a mechanism underlying macrosomia and prolonged pregnancy.

Persistently elevated troponin I in paracetamol hepatotoxicity: association with liver injury, organ failure, and outcome.

CONTEXT: An elevated troponin I (TnI) is associated with a poorer prognosis during critical illness. OBJECTIVE: Our aims were to determine whether significant paracetamol-induced hepatotoxicity was associated with an elevated TnI; if this elevation was persistent and was associated with worse clinical outcomes. MATERIALS AND METHODS: In this retrospective cohort study, the requirement for orthotopic liver transplantation (OLT) or death and/or the development of multiorgan failure (MOF) was evaluated for 48 consecutive patients admitted to the Royal Infirmary of Edinburgh (a university tertiary referral centre) with acute liver injury or acute liver failure secondary to paracetamol overdose. RESULTS: TnI was elevated (≥ 0.05 ng/L) in 13/48 patients (27%). This appeared to be sustained for at least 6 days which has not been shown previously in the context of Acute Liver Injury (ALI). Elevated TnI was strongly associated with MOF, with the requirement for inotropic support being the strongest predictor (p = 0.003, OR 9.00, 95% CI 2.13-37.98). TnI elevations also correlated strongly with Acute Physiology and Chronic Health Evaluation (APACHE) II scores (p = 0.0006, r = 0.482, 95% CI 0.22-0.68) and with interleukin 6 (IL-6) levels (p = 0.0001, r = 0.55, 95% CI 0.29-0.73). Although a raised TnI was associated with a markedly increased risk of death or orthotopic liver transplant (p = 0.005, OR 7.73, 95% CI 1.87-32.05) on univariate analysis, this was primarily seen in the context of MOF (SOFA score p = 0.003, OR 1.23, 95% CI 1.07-1.41) and was not an independent predictor of death. There was no correlation between TnI or outcome with other cardiac biomarkers and markers of cardiovascular risk. DISCUSSION AND CONCLUSION: An elevated TnI in the context of acute liver injury or liver failure following paracetamol overdose is associated with a significantly worse patient outcome but it is not an independent prognostic factor. Further studies should be undertaken to investigate the mechanism behind this elevated troponin association.

Predictors of treatment failure, incipient hypothyroidism, and weight gain following radioiodine therapy for Graves' thyrotoxicosis.

BACKGROUND: Following radioiodine ((131)I) therapy, both late recognition of hypothyroidism and treatment failure may result in adverse outcomes. AIM: We sought to assess indicators of both incipient hypothyroidism and treatment failure following (131)I and determine factors predictive of weight gain. SUBJECTS AND METHODS: Retrospective study of 288 patients receiving (131)I for treatment of Graves' thyrotoxicosis. Primary outcome measures were thyroid status and weight change at 1 yr following (131)I. RESULTS: The treatment failure rate at 1 yr was 13.5%. Hypothyroidism developed in 80.9%, with 58.5% of patients having levels of free T4 (fT4) <6 pmol/l at diagnosis. Patients receiving thionamides before and after (131)I had significantly higher levels of treatment failure (23.3%) than those with no thionamide exposure (6.3%, p=0.003), but also had more active Graves' disease. Following (131)I, development of a detectable TSH or low-normal fT4 levels was not associated with recurrent thyrotoxicosis. Median weight gain was 5.3 kg, although patients with nadir fT4 levels <6 pmol/l gained an average 2 kg more than those with levels >6 pmol/l (p=0.05). The main predictor of weight gain was fT4 level immediately prior to treatment; those in the lowest tertile gained a median 3.1 kg whilst those in the highest tertile gained 7.4 kg (median difference 4.3 kg; 95% confidence interval: 2.5-6.2). CONCLUSIONS: Marked hypothyroidism following (131)I is common and often occurs early. Simple biochemical parameters may help identify incipient hypothyroidism and potentially limit excess weight gain. Treatment failure is common in patients with severe thyrotoxicosis and in such cases larger doses of (131)I may be warranted.

Effects of selenium compounds on induction of DNA damage by broadband ultraviolet radiation in human keratinocytes.

Background Ultraviolet radiation (UVR), a ubiquitous environmental genotoxin for the skin, produces DNA damage. The trace element selenium induces synthesis of the glutathione peroxidase and thioredoxin reductase enzyme families. These selenoenzymes detoxify a range of toxic compounds generated by free radicals. Objectives To assess the effects of pretreatment of primary human keratinocytes with selenium on UVR-induced DNA damage. Methods Cells were irradiated with UVR from FS-20 lamps and were subjected to comet assay. Results Comet tail length due to UVR-induced T4 endonuclease V-sensitive sites (caused by cyclopyrimidine dimers, CPDs) increased to 35 +/- 4.5 microm (mean +/- SD) immediately after irradiation (time 0 h, 100%). After 4 h, 68% of the damage remained and after 24 h, 23% of the damage was still present. Treatment with up to 200 nmol L-1 selenomethionine or 50 nmol L-1 sodium selenite had no effect on CPD formation or rates of repair, or on the number of excision repair sites as measured by cytosine arabino furanoside and hydroxyurea treatment. However, selenite and selenomethionine protected against oxidative damage to DNA as measured by formation of formamidopyrimidine (FaPy) glycosylase-sensitive sites, which are indicative of 8-hydroxy-2-deoxyguanosine photoproduct formation. In this assay, irradiation of keratinocytes increased mean +/- SD glycosylase-specific comet tail length from 5 +/- 1.5 microm to 19 +/- 3.3 microm. Preincubation for 18 h with 50 nmol L-1 selenite abolished the UVR-induced increase in comet length. Preincubation with 200 nmol L-1 selenomethionine was similarly protective. Conclusions Selenite and selenomethionine protect keratinocytes from UVR-induced oxidative damage, but not from formation of UVR-induced excision repair sites.

Selenium protects primary human keratinocytes from apoptosis induced by exposure to ultraviolet radiation.

The generation of reactive oxygen species has been implicated in ultraviolet radiation (UVR)-induced skin damage. In mice, increasing dietary selenium intake protects skin from UVR-induced DNA damage and photocarcinogenesis. We sought to determine whether selenium supplementation could protect keratinocytes from apoptosis resulting from exposure to broadband (TL20W/12) UVR. Unirradiated cultures contained 6.5 +/- 1% apoptotic cells; the maximum percentage of apoptotic cells (34 +/- 5%) was seen 16 h after UVR of 600 J/m(2). Under these conditions cell death from necrosis was 15 +/- 2.5% of the total cells. A 24-h preincubation with sodium selenite (10 nm(-1) microm) or selenomethionine (50 nm(-1) microm) protected cultured human keratinocytes from UVR-induced apoptosis. In primary keratinocytes the greatest reduction in apoptosis was found with 100 nm of either selenium compound (71% reduction in the numbers of total apoptotic cells; P < 0.01). Supplementation with 100-200 nm selenite or selenomethionine prevented UVR-induced apoptosis, but did not decrease the levels of UVR-induced p53, as measured by Western blotting. Collectively, this data suggests that selenium prevents UVR-induced cell death by inhibiting p53-independent cell death pathways.

Selenoprotein expression in endothelial cells from different human vasculature and species.

Selenium (Se) can protect endothelial cells (EC) from oxidative damage by altering the expression of selenoproteins with antioxidant function such as cytoplasmic glutathione peroxidase (cyGPX), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and thioredoxin reductase (TR). If the role of Se on EC function is to be studied, it is essential that a model system be chosen which reflects selenoprotein expression in human EC derived from vessels prone to developing atheroma. We have used [75Se]-selenite labelling and selenoenzyme measurements to compare the selenoproteins expressed by cultures of EC isolated from different human vasculature with EC bovine and porcine aorta. Only small differences were observed in selenoprotein expression and activity in EC originating from human coronary artery, human umbilical vein (HUVEC), human umbilical artery and the human EC line EAhy926. The selenoprotein profile in HUVEC was consistent over eight passages and HUVEC isolated from four cords also showed little variability. In contrast, EC isolated from pig and bovine aorta showed marked differences in selenoprotein expression when compared to human cells. This study firmly establishes the suitability and consistency of using HUVEC (and possibly the human cell line EAhy926) as a model to study the effects of Se on EC function in relation to atheroma development in the coronary artery. Bovine or porcine EC appear to be an inappropriate model.

Myocardial infarction redefined: the new ACC/ESC definition, based on cardiac troponin, increases the apparent incidence of infarction.

OBJECTIVES: To investigate the impact of the redefinition of the diagnostic criteria for myocardial infarction on its apparent incidence in a non-selected and representative series of patients admitted with acute chest pain. DESIGN: Single centre prospective study. SETTING: Medical assessment unit and cardiology wards of an inner city university hospital. PATIENTS: 80 consecutive patients aged over 25 years admitted with suspected ischaemic acute chest pain (excluding those where the ECG indicated definite myocardial infarction). INTERVENTIONS: Measurement of concentrations of conventional cardiac biomarkers (creatine kinase and its MB isoenzyme, CK-MB) and concentrations of the highly specific diagnostic indicator of myocardial damage, cardiac troponin I (cTnI) 12-24 hours after the onset of acute chest pain. MAIN OUTCOME MEASURES: Frequency of myocardial infarction as assessed by conventional diagnostic criteria (creatine kinase and CK-MB) plus clinical symptoms of infarction, versus frequency of infarction based on high sensitivity troponin assays. RESULTS: Among patients with acute coronary syndromes but non-diagnostic ECG changes, 40% (32/80) fulfilled the new criteria for myocardial infarction using high sensitivity cTnI measurement, compared with 29% (23/80) using the conventional diagnostic criteria for myocardial infarction. CONCLUSIONS: The implications of the redefinition of myocardial infarction on patients, their care, and the use of health care resources are substantial.

Inhibition of ultraviolet B radiation-induced interleukin 10 expression in murine keratinocytes by selenium compounds.

BACKGROUND: Selenium is an essential trace nutrient necessary for the normal function of the immune system. Selenium compounds protect mice against ultraviolet (UV) B-induced tumours, probably by preventing oxidative damage to the host skin cells and to the host immune system. One possible mechanism of protection is that selenium can prevent oxidative stress-induced release of cytokines such as interleukin (IL)-10, which could suppress cell-mediated immunity. OBJECTIVES: To determine whether selenium compounds can inhibit UVB induction of IL-10 protein in murine keratinocytes. METHODS: The murine keratinocyte cell line PAM 212 was treated with or without selenomethionine (50-200 nmol L-1) or sodium selenite (1-50 nmol L(-1)) for 24 h before exposure to 200 J m(-2) UVB. The cells were stained with an antibody to IL-10, 24 h after irradiation. RESULTS: Preincubation with both selenium compounds inhibited UVB induction of IL-10 immunostaining, although selenomethionine was more effective. Pretreatment with 200 nmol L(-1) selenomethionine decreased IL-10 immunostaining to levels seen in the unirradiated controls. CONCLUSIONS: The protective effects of selenium against UVB-induced skin cancer in murine models may result, in part, from its ability to inhibit release of cytokines that are capable of suppressing cell-mediated immunity.

Effect of spinal anaesthesia on plasma concentrations of glutathione S-transferase.

BACKGROUND: Plasma glutathione S-transferase (GST) concentration measurement is a sensitive and specific index of hepatocellular injury. GST concentration increases after anaesthesia with most volatile anaesthetic agents, but not after propofol. Such increases are thought to result from reduced liver blood flow. The effect on GST concentration of spinal (subarachnoid) anaesthesia, which might also reduce liver blood flow, is not known. METHODS: We studied the effects of spinal anaesthesia on GST concentrations measured by specific radioimmunoassay in 33 patients undergoing intermediate orthopaedic, general or gynaecological surgery. GST concentrations were measured before anaesthesia and 3, 6 and 24 h after induction of anaesthesia. Hypotension (systolic blood pressure <70% of pre-induction value) was rapidly corrected with i.v. ephedrine. RESULTS: Mean duration of surgery was 41 min (range 11-80). No increase in GST concentration was observed at any time, but at 24 h GST concentration was significantly reduced (P<0.05). One patient in whom hypotension was not treated developed a greatly increased GST concentration at 3 h. CONCLUSION: We found no association between spinal anaesthesia and disturbance of hepatocellular integrity when hypotension does not occur or is rapidly corrected.

Selenium status of cats in four regions of the world and comparison with reported incidence of hyperthyroidism in cats in those regions.

OBJECTIVE: To assess selenium (Se) status of cats in 4 regions of the world and to compare results for Se status with reported incidence of hyperthyroidism in cats in those regions. ANIMALS: 50 cats (30 from 2 regions with an allegedly high incidence of hyperthyroidism and 20 from 2 regions in which the disease is less commonly reported). PROCEDURE: Hematologic samples (heparinized whole blood, plasma, and RBC fractions) were obtained from 43 healthy euthyroid cats and 7 hyperthyroid cats. Plasma concentration of Se and activity of glutathione peroxidase (GPX) in whole blood and plasma were determined. RESULTS: Plasma concentration of Se and GPX activity in whole blood or plasma did not differ significantly among cats from the 4 regions. However, cats had a plasma concentration of Se that was approximately 5 times the concentration reported in rats and humans. The GPX activity in whole blood or plasma in cats generally was higher than values reported in rats or humans. CONCLUSIONS AND CLINICAL RELEVANCE: Cats have higher Se concentrations in plasma, compared with values for other species. However, Se status alone does not appear to affect the incidence of hyperthyroidism in cats. High Se concentrations may have implications for health of cats if such concentrations are influenced by the amount of that micronutrient included in diets.

Thioredoxin reductase and cytoplasmic glutathione peroxidase activity in human foetal and neonatal liver.

Cytosolic thioredoxin reductase (TR) is an FAD-containing homodimeric selenoenzyme which, together with thioredoxin (Trx) and NADPH, forms a powerful oxidoreductase system. Cytoplasmic glutathione peroxidase (GPX-1) is a selenoprotein with antioxidant activity. The TR/Trx system has been associated with cellular processes including regulation of cell growth, and modification of activity of transcription factors. TR may also act as an antioxidant. We have measured TR activity, TR concentration, and GPX-1 activity in human hepatic cytosols from foetuses and neonates. The concentration of TR was significantly greater (P<0.05) in foetal (43.6, 37.9-50.8 microg/g protein, median, interquartile range) than in neonatal liver (11.6, 8.70-15.0 microg/g). This was also true of TR activity which was 2.1, 1.8-2.5 U/g protein in foetal, and 0.65, 0.44-0.74 U/g protein in neonatal liver (P<0.0005). Similarly, GPX-1 activity was significantly higher (P<0.005) in the foetal (199.7, 144.0-227.9 U/g protein) than in neonatal (77.0, 58.4-110.3 U/g protein) hepatic cytosol. Overall, foetal liver expressed approx. 3-fold higher activities of TR and GPX-1 than neonatal liver.

Selenite protects human endothelial cells from oxidative damage and induces thioredoxin reductase.

The ability of selenium to protect cultured human coronary artery endothelial cells (HCAEC), human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) from oxidative damage induced by 100 microM t-butyl hydroperoxide (t-BuOOH) was compared. Preincubation of human endothelial cells for 24 h with sodium selenite at concentrations as low as 5 nM provided significant protection against the harmful effects of 100 microM t-BuOOH, with complete protection being achieved with 40 nM selenite. The preincubation period was required for selenite to exert this protective effect on endothelial cells. When compared with selenium-deficient cells, the activities of cytoplasmic glutathione peroxidase (GPX-1), phospholipid hydroperoxide glutathione peroxidase (GPX-4) and thioredoxin reductase (TR) were each induced approx. 3--4-fold by 40 nM selenite. HCAEC and HUVEC showed great similarity in their relative abilities to resist oxidative damage in the presence and absence of selenite, and the activities of TR and the GPXs were also similar in these cell types. BAEC were more susceptible to damage by 100 microM t-BuOOH than were human endothelial cells, and could not be protected completely by incubation with selenite at concentrations up to 160 nM. The activity of TR in human endothelial cells was approx. 25-fold greater than that in BAEC of a similar selenium status, but GPX-1 and GPX-4 activities were not significantly different between the human and bovine cells. These studies, although performed with a small number of cultures, show for the first time that selenium at low doses can provide significant protection of the human coronary artery endothelium against damage by oxidative stress. TR may be an important antioxidant selenoprotein in this regard, in addition to the GPXs. The data also suggest that HUVEC, but not BAEC, represent a suitable model system in which to study the effects of selenium on the endothelium of human coronary arteries.

Thioredoxin alters the matrix metalloproteinase/tissue inhibitors of metalloproteinase balance and stimulates human SK-N-SH neuroblastoma cell invasion.

Thioredoxin (Trx) inhibited tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 activity with an approximate IC50 of 0.3 microM, matrix metalloproteinase (MMP)-2 activity with an approximate IC50 of 2 microM but did not inhibit MMP-9 activity. This differential capacity of Trx to inhibit TIMP and MMP activity resulted in the promotion of MMP-2 and MMP-9 activity in the presence of molar TIMP excess. Inhibition of TIMP and MMP-2 activity by Trx was dependent upon thioredoxin reductase (TrxR), was abolished by Trx catalytic site mutation and did not result from TIMP or MMP-2 degradation. HepG2 hepatocellular carcinoma cells induced to secrete Trx inhibited TIMP activity in the presence of TrxR. SK-N-SH neuroblastoma cells secreted TrxR, which inhibited TIMP and MMP-2 activity in the presence of Trx. Trx stimulated SK-N-SH invasive capacity in vitro in the absence of exogenous TrxR. This study therefore identifies a novel extracellular role for the thioredoxin/thioredoxin reductase redox system in the differential inhibition of TIMP and MMP activity and provides a novel mechanism for altering the TIMP/MMP balance that is of potential relevance to tumor invasion.

Effects of organic and inorganic selenium supplementation on selenoenzyme activity in blood lymphocytes, granulocytes, platelets and erythrocytes.

The blood selenium (Se) concentration in the U.K. population has declined by approx. 50% between 1974 and 1991, reflecting a large decrease in dietary Se supply, with intakes only half the reference nutrient intake of 1 microg/kg body weight. Tissue levels of Se are readily influenced by dietary intake. Therefore selenoprotein activity may be sub-optimal due to low Se status, and thus compromise normal cell function. To examine the effects of changing Se intake on selenoproteins, we have determined the relative effectiveness of organic selenomethionine and inorganic sodium selenite (50 microg of Se daily for 28 days) in modulating glutathione peroxidase activities in blood cells from 45 healthy men and women, from a U.K. population. Transient and acute changes in lymphocyte, granulocyte and platelet phospholipid-hydroperoxide glutathione peroxidase (GPx4) activity occurred by day 7 or 14 of sodium selenite treatment and by day 7 in lymphocytes from selenomethionine-treated subjects compared with controls taking a placebo. In contrast, GPx4 activity in granulocytes and platelets in the selenomethionine group increased gradually over the 28 days. Cytosolic glutathione peroxidase (GPx1) activity in these blood cells from both treatment groups increased gradually over the 28 days. For each cellular selenoenzyme activity a significant inter-individual difference (P<0.001) in the extent of the response to Se supplementation was observed, but this was not related to blood Se concentrations either before or after treatments. Significant inverse correlations were evident between baseline enzyme activities and percentage change in activity after 28 days of supplementation [e.g. lymphocyte GPx4, r=-0.695 (P<0.001)], indicating that pre-treatment activity may be sub-optimal as a result of poor Se status. The different and contrasting effects that Se supplementation had on blood selenoenzyme activities may be indicative of a difference in metabolic need for Se regulated at the level of Se-dependent cell function.

Thyroid hormone deiodination in the domestic cat.

We have investigated thyroid hormone deiodination in the liver, kidney and thyroid of the domestic cat. Affinity labelling with (125)I-bromoacetyl reverse T(3) (125)(I-BrAc-rT(3) demonstrated that liver and kidney, but not the thyroid, express type I iodothyronine deiodinase (IDI), results that were confirmed by measuring the activity of the IDI using (125)I-rT(3) and T(4) as substrate. Feline hepatic and renal IDI metabolised rT(3) at approximately 0.2% of the rate of rat hepatic IDI under identical assay conditions. The K(m) of the feline enzyme was at least 500-fold greater than that of rat IDI. However, feline and rat hepatic IDI metabolised T(4) at a similar rate and had similar K(m) values (1.35 microM and 2.25 microM, respectively). This study demonstrates that cats and rats express IDI in the liver and kidney in similar concentrations; however, the feline enzyme appears unable to utilise rT(3) as a substrate under physiological conditions.

Serum thyroxine binding capacity-dependent bias in an automated free thyroxine assay.

The magnitude of serum thyroxine (T4) binding capacity (sBC) dependent bias in the AXSYM free thyroxine (FT4) assay was assessed using two recently described tests. One of the tests uses a direct equilibrium dialysis (ED) FT4 assay as the reference method. The results obtained with the AXSYM method were compared with those obtained by the ED FT4 method in patient sera having a wide range of sBC. The other test involves comparison of the FT4 results obtained following dilution of sera by an inert buffer, to theoretically derived FT4 results. As serum dilution causes a predictable decrease in sBC, the demonstration of a negative bias whose magnitude increases in parallel to the dilution, is indicative of an sBC-dependent bias. The AXSYM FT4 assay exhibited a significant sBC-dependent bias. This sBC-dependent bias is likely to have been caused by the presence of significant amounts of T4 binding proteins in the assay reagents.

Thioredoxin reductase is the major selenoprotein expressed in human umbilical-vein endothelial cells and is regulated by protein kinase C.

Damage to the endothelium by reactive oxygen species favours atherogenesis. Such damage can be prevented by selenium, which is thought to exert its actions through the expression of selenoproteins. The family of glutathione peroxidases (GPXs) may have antioxidant roles in the endothelium but other intracellular and extracellular selenoproteins with antioxidant actions may also be important. The selenoproteins expressed by cultured human umbilical-vein endothelial cells (HUVECs) were labelled with [(75)Se]selenite and separated using SDS/PAGE. HUVECs secreted no extracellular selenoproteins. There were distinct differences between the intracellular selenoprotein profile of (75)Se-labelled HUVECs and those of other tissues. A single selenoprotein with a molecular mass of 58 kDa accounted for approx. 43% of the intracellular (75)Se-labelled proteins in HUVECs. This protein was identified by Western blotting as the redox-active lipid-hydroperoxide-detoxifying selenoprotein, thioredoxin reductase (TR). TR expression in HUVECs was down-regulated by transiently exposing cells to the phorbol ester PMA for periods as short as 1 min. However, there was a delay of 48 h after PMA exposure before maximal down-regulation of TR was observed. The protein kinase C (PKC) inhibitor bisindolylmaleimide I hydrochloride had no effect on TR expression when added alone, but the agent prevented the down-regulation of TR expression seen with PMA. The calcium ionophore A23187 increased TR expression in HUVECs after a 12-h exposure, but the maximal effect was only observed after a 35-h exposure. These findings suggest that TR may be an important factor in the known ability of Se to protect HUVECs from peroxidative damage. Furthermore, the results also suggest that TR expression can be negatively regulated through PKC. It is possible that TR expression may be positively regulated by the calcium-signalling cascade, although TR induction by A23187 may be due to toxicity.