Metallothionein isoform expression by breast cancer cells.

Expression of metallothionein (MT) isoforms by a human breast cancer cell line, PMC42, which retains many characteristics of normal breast epithelial cells and expresses functional estrogen receptors, was examined because it has been proposed that human breast cancer cells which are estrogen receptor positive can be differentiated from those which are estrogen receptor negative, by failure to express MT-1E [J.A. Friedline, S.H. Garrett, S. Somji, J.H. Todd, D. A. Sens, Differential expression of the MT-1E gene in estrogen-receptor positive and -negative breast cancer cell lines, Am. J. Pathol. 152 (1998) 23-27]. Using RT-PCR, PMC42 cells were found to transcribe genes for the MT isoforms IE, IX and 2A but not 1A or 1H. In order to examine which of the expressed isoforms might protect against metal toxicity, the cells were challenged with high concentrations of zinc and copper. Using competitive RT-PCR, cells resistant to 500 microM zinc showed 7+/-2 fold (SD, n=3) increases in expression of MT-1X and 6+/-3 fold increases in expression of MT-2A compared to control cells in normal media. For cells resistant to 250 microM copper the corresponding increases were 37+/-13 and 60+/-20 fold, whilst for control cells treated with 250 microM copper for only 6 h, increases were 10+/-3 and 6+/-3 fold. There was only a low level of expression of MT-1E in untreated cells and but a >120 fold increase in copper- resistant cells. Thus estrogen receptor positive cells cannot, in general, be differentiated from estrogen receptor negative cells by failure to express MT-1E, as suggested by Friedline et al. (1998). Increased expression of MT-1E, as well as MT-1X and MT-2A, protects against metal toxicity in PMC42 breast cancer cells.

Expression of Menkes disease gene in mammary carcinoma cells.

Two P-type ATPases, MNK and WND were recently shown to be defective in the human disorders of copper transport, Menkes disease and Wilson disease respectively. These proteins are important in copper homeostasis but their full physiological function has not been established. This study uses the human breast carcinoma line, PMC42, to investigate copper transport in the mammary gland. Northern blot analysis indicated that both MNK and WND mRNA are expressed in these cells. Western blot analysis with an MNK-specific antibody demonstrated a band of approx. 178 kDa, close to the expected size of 163 kDa. Treatment of PMC42 cells with lactational hormones (oestrogen and progesterone for 3 days followed by dexamethasone, insulin and prolactin for a further 3 days) did not produce an obvious increase in MNK expression as measured by Northern and Western blots. By using indirect immunofluorescence with the MNK antibody, the intracellular distribution of MNK was found to be predominantly perinuclear, consistent with Golgi localization. Punctate staining was also seen in a smaller proportion of cells, suggesting that some MNK is associated with endosomes. Treatment of PMC42 cells with lactational hormones increased the intensity of the perinuclear and punctate fluorescence. Exposure of cells to 100 mM copper resulted in the dispersion of the fluorescence towards the periphery of the cell. The results suggest a role for MNK in the secretion of copper into milk and that PMC42 cells are a valuable model for investigating the detailed cellular function of MNK and WND.

Phospholipase D activation by P2Z-purinoceptor agonists in human lymphocytes is dependent on bivalent cation influx.

The role of bivalent cations in ATP-stimulated phospholipase D (PLD) activity was investigated in human leukaemic lymphocytes. Cells were labelled with [3H]oleic acid and incubated with extracellular ATP or benzoylbenzoic ATP in the presence of 1 mM Ca2+ and butanol, and PLD activity was assayed by the accumulation of [3H]phosphatidylbutanol ([3H]PBut). ATP stimulated PLD activity in a dose-dependent manner, and the inhibitory effects of suramin, oxidized ATP and extracellular Mg2+ suggested that the effect of ATP was mediated by P2Z purinoceptors known to be present on lymphocytes. Thapsigargin increased cytosolic [Ca2+] but did not stimulate PLD activity, whereas preloading cells with a Ca2+ chelator reduced cytosolic [Ca2+] and, paradoxically, potentiated ATP-stimulated [3H]PBut accumulation. ATP-stimulated [3H]PBut formation was supported by both Ba2+ and Sr2+ when they were substituted for extracellular Ca2+. Addition of EGTA to block bivalent cation influx inhibited the majority of ATP-stimulated PLD activity. Furthermore ATP-stimulated PLD activity showed a linear relationship to extracellular [Ba2+], and ATP-induced 133Ba2+ influx also had a linear dependence on extracellular [Ba2+]. These results suggest that ATP stimulates PLD activity in direct proportion to the influx of bivalent cations through the P2Z-purinoceptor ion channel and that this PLD activity is insensitive to changes in bulk cytosolic [Ca2+].

Responses of sheep and kitten isolated coronary arteries to some vasoactive agents.

In the present study, the effects of a range of vasoactive agents were assessed in sheep isolated coronary artery strips and in kitten isolated perfused coronary arteries. Both preparations contained muscarinic cholinoceptors. However, these receptors mediated contraction of sheep coronaries while dilatation was seen in the kitten. In sheep arteries, 5-hydroxytryptamine caused increases in resting tone and relaxation of acetylcholine-induced tone, while vasoconstriction was the predominant response in the kitten. Angiotensin increased tone in both preparations, although tachyphylaxis was evident. Conversely, adenosine and ATP caused reductions in tone in both preparations. Alpha-adrenoceptor agonists caused vasodilatation in kitten coronaries and decreased acetylcholine-induced tone in sheep arteries. However, weak contractions were observed in sheep coronaries in the absence of induced tone. Results also indicate that sympatomimetic amines mediate relaxation of sheep coronary arteries via beta 1-adrenoceptors. Sheep coronaries are readily and inexpensively obtained and provide a useful tool for the further study of coronary vascular smooth muscle.

Potentiating effect of indomethacin on relaxations of sheep isolated coronary artery caused by sympathetic nerve stimulation or exogenous noradrenaline.

Indomethacin, 2.8 microM, potentiated relaxation responses of sheep, isolated, superfused coronary arteries caused by electrical transmural stimulation of sympathetic nerves by 2.3 fold, while responses to exogenous noradrenaline were not significantly altered. A ten fold higher concentration of indomethacin potentiated responses to sympathetic nerve stimulation by 3.4 fold and to exogenous noradrenaline by 1.8 fold. Stimulation-induced efflux of radioactivity derived from 3H-(-)-noradrenaline in sheep coronary arteries was not significantly altered by indomethacin (28 microM). Thus, while indomethacin-induced potentiation of relaxation responses to sympathetic nerve stimulation, was not due to an inhibition of prejunctional actions of prostaglandins, it may involve inhibition of the synthesis of prostaglandins which have post-junctional effects.

Effect of indomethacin on responses of sheep isolated coronary artery to arachidonic acid.

Indomethacin (2.8 mumol/l) did not consistently affect basal tone of sheep coronary artery strips, while a ten-fold higher concentration increased tension in 50% of the preparations tested. When acetylcholine was used as a spasmogen, oscillations in induced tone and relaxations produced by arachidonic acid (6.6 mumol/l) were abolished by indomethacin, 2.8 mumol/l and 7 mumol/l, respectively. Prostacyclin (PGI2) and prostaglandin E1 decreased and PGE2 increased arterial tension while PGF2 alpha was inactive. Responses to PGI2 were reduced by indomethacin (28 mumol/l) but not by indomethacin (2.8 mumol/l). It is suggested that sheep isolated coronary arteries synthesize and release prostacyclin in the presence of acetylcholine and arachidonic acid and that such synthesis can be inhibited by indomethacin.

Metabolism of [3H]-(+/-)-isoprenaline by isolated atria and coronary arteries of the kitten.

1 Isolated coronary arteries of the kitten accumulated more unchanged isoprenaline and metabolized more amine than atria following incubation for 1 to 20 min with [3H]-(+/-)-isoprenaline (25 ng/ml or 5 micrograms/ml). 2 Cortisol (10 or 80 microM), U-0521 (120 microM) and oxytetracycline (100 microM) all reduced metabolite formation. 3 Cortisol inhibited 'Iso InfluxMin' (cellular isoprenaline accumulation plus total metabolite production). In contrast, it increased, decreased or did not alter accumulation of unmetabolized isoprenaline, depending upon the experimental conditions. 4 Isoprenaline accumulation was increased in atria and reduced in coronary arteries by U-0521, while oxytetracycline reduced accumulation in coronary arteries at the high amine concentration. 5 It is concluded that in atria, cortisol inhibits metabolism and has differential effects on a number of extraneuronal compartments which accumulate isoprenaline. Both cortisol and U-0521 appear to be extraneuronal uptake inhibitors and inhibitors of catechol-O-methyltransferase in coronary arteries. Oxytetracycline may have effects additional to inhibition of isoprenaline binding to connective tissue fibres.

Effects of uptake inhibitors on responses of sheep coronary arteries to catecholamines and sympathetic nerve stimulation.

1. Transmural stimulation of intrinsic sympathetic nerves and exogenous catecholamines produce beta 1-adrenoceptor mediated relaxant responses in strips of contracted sheep coronary artery. 2. The neuronal uptake inhibitors, metaraminol, cocaine and desipramine and the extraneuronal uptake inhibitor, cortisol, failed to potentiate responses to noradrenaline or sympathetic stimulation; responses to isoprenaline were enhanced by cortisol. 3. Oxytetracycline, which inhibits binding to connective tissue fibres, did not affect responses to noradrenaline or nerve stimulation. 4. 17 beta-Oestradiol, caffeine and U0521 proved to be unsuitable compounds for studying catecholamine inactivation since they non-selectively potentiated responses to noradrenaline and isoprenaline. 5. It is concluded that catecholamine inactivation processes do not modify transmitter function in sheep coronary arteries.

Metabolic effects of oral contraceptives containing 30 micrograms and 50 micrograms of oestrogen.

In Study A, biochemical data from 17 women who were not taking oral contraceptives were compared with those from women taking preparations which contained either 30 microgram of ethinyl oestradiol and 150 microgram of D-norgestrel (18 women) or 50 microgram of ethinyl oestradiol and 250 microgram of D-norgestrel (nine women). In Study B, biochemical data were collected from eight women before and during the first three or four months therapy with preparations containing 30 microgram of ethinyl oestradiol and 150 microgram of D-norgestrel. The two oral contraceptive dosage forms studied produced qualitatively and quantitatively similar metabolic changes. Both caused an increase in serum concentration of triglycerides (30% to 33%), beta-lipoproteins (27% to 29%) and ceruloplasmin (75% to 90%), and a decrease in serum levels of antithrombin III (22% to 29%) and ascorbic acid (30% to 42%). Serum cholesterol and phospholipid concentrations were unchanged. However, the proportion of serum cholesterol carried by alpha-lipoproteins (high density lipoproteins) decreased, while that carried by beta-lipoproteins (low density and very low density lipoproteins) increased. The former change is in the same direction, but much smaller than that observed in coronary heart disease.

PIP: 2 studies, A and B, were conducted to determine the metabolic effects of oral contraceptives (OCs). The subjects were healthy nonsmokers (17 to 27 years old, 1.52 m. to 1.75 m. in height, and 50 kg. to 60 kg. in weight) who were not undergoing any therapy. Controls did not have any previous history of hormonal therapy. In Study A, biochemical data from 17 women who were not on OCs were compared with those of women taking pills containing either 30 ug of ethinyl estradiol (EE) and 150 ug of D. norgestrel (18 women) or 50 ug. of EE and 250 ug. of D. norgestrel (9 women). In Study B, 8 women were studied before and during 3 to 4 cycles of low-dosed OC therapy (30 ug. of EE and 150 ug. of D. norgestrel). In both studies, the 2 oral contraceptive dosage forms had similar metabolic quantitative and qualitative changes: both resulted in an increase in serum concentration of triglycerides (30 to 33%), B-lipoproteins (27 to 29%), and ceruloplasmin (75 to 90%), and a decrease in serum levels of antithrombin 3 (22 to 29%) and ascorbic acid (30 to 42%). Serum cholesterol and phospholipid concentration did not change. The proportion of serum cholesterol carried by a-lipoproteins (high density lipoproteins) did decrease (the change is much smaller than that seen in coronary heart disease) while that carried by B-lipoproteins (or low density and very low density lipoproteins) increased. A significant negative correlation was observed between serum concentrations of ascorbic acid and cholesterol; this is interesting as clinical and experimental evidence suggests that latent ascorbic acid deficiency leads to hypercholesteroleamia, while ascorbic acid supplements reduce plasma cholesterol levels. As 500 mg. daily of ascorbic acid supplements supposedly help maintain normal ascorbic acid levels in blood during OC use, they should perhaps be prescribed to pill users in this study.

Catecholamine uptake by isolated coronary arteries and atria of the kitten.

Kitten coronary arteries and atria incubated with [3H]-(-)-noradrenaline or [3H]-(+/-)-isoprenaline showed concentration- and temperature-dependent accumulation of amines (unchanged and/or as metabolites). In addition, coronary arteries incubated with [3H]-(+/-)-isoprenaline showed temperature-insensitive amine accumulation due to binding to connective tissue fibres. 2 With low concentrations of [3H]-(-)-noradrenaline (20 ng/ml) accumulation of radioactivity in both tissues was neuronal since it was reduced by desipramine, metaraminol or cocaine but not by the extraneuronal inhibitor, cortisol. 3 In coronary arteries incubated with [3H]-(+/-)-isoprenaline (5 microgram/ml) for 1 or 10 min, uptake was extraneuronal since it was inhibited by cortisol, 17beta-oestradiol or phenoxybenzamine. 4 Atria incubated with (+/-)-isoprenaline (5 to 1000 microgram/ml) showed non-neuronal, biphasic accumulation of amine. There was a high capacity, low affinity initial uptake process (apparent Km 136 micron) which was resistant to steroidal extraneuronal uptake inhibitors but which could be abolished by phenoxybenzamine. A slower uptake occurred after 2 min which was sensitive to steroidal and other extraneuronal uptake inhibitors. 5 Inhibition of uptake processes did not alter sensitivity of coronary arteries to dilator effects of catecholamines. However, experiments with extraneuronal uptake inhibitors were limited by the relaxant effects of cortisol and 17beta-oestradiol themselves. 6 In atria inhibition of neuronal uptake by desipramine or cocaine led to supersensitivity to (-)-noradrenaline but not to (-)-adrenaline or (+/-)-isoprenaline. Chronotropic responses to (-)-noradrenaline were increased five fold and inotropic responses three fold.

Independence of oral contraceptive-induced changes in glucose tolerance and plasma cortisol levels.

1. Following a 50 g oral glucose load, plasma glucose concentrations and plasma cortisol concentrations were measured in twenty-five young healthy women. 2. When compared to controls, oral contraceptive users (OC-users) were found to show a small but significant decrease in glucose tolerance while plasma cortisol values were increased more than two-fold. 3. The decreased glucose tolerance in OC-users did not show a significant correlation with raised plasma cortisol levels.

Letter: Pyridoxine and oestrogen-induced glucose intolerance.

PIP: Clinical examination of the possibility that xanthurenic acid is the cause of oral contraceptive (OC) induced glucose tolerance and gestational diabetes has been carried out. The reduction by pyridoxine of excessive xanthurenic acid excretion following a tryptophan load has been associated with an improvement in glucose tolerance, yet it is not believed that the 2 are causally related. Instead, the beneficial effects of pyridoxine are possibly because of normalization of other tryptophan metabolites. Some women suffer from estrogen-induced glucose intolerance independent of any abnormalities in xanthurenic acid excretion. It has been found that the administration of xanthurenic acid 120 mg/kg intraperitoneally in ammoniated saline failed to alter blood glucose in male Sprague Dawley or Wistar rats. The experiments were repeated using the special high fat diet which had been used in the original studies on the diabetogenic effects of xanthurenic acid, and xanthurenic acid was found to be inactive. Xanthurenic acid did not impair glucose tolerance when given 1 hour before an oral glucose load in further experiments. The failure to demonstrate any diabetogenic effect of xanthurenic acid could not be because of contamination with the 8-methyl ether of xanthurenic acid, which has been reported to be antidiabetogenic, since the sample of xanthurenic acid used was found to be free from this contaminant.^ieng

Effects of changes in external calcium concentration and frequency of stimulation on inotropic responses to isoprenaline and tolbutamide in rabbit atria.

1. Driven left atrial preparations were used to study inotropic responses to isoprenaline and tolbutamide. 2. Decreasing the external calcium concentration from 2.2 to 1.1 and 0.55 mM decreased resting force of contraction but increased the maximal inotropic responses to isoprenaline. There was an associated increase in EC50 values. 3. Decreasing the external calcium concentration from 2.2 to 1.1 mM also increased the maximal inotropic responses to tolbutamide. These responses were 23- and 44-fold less than those to isoprenaline at equivalent calcium concentrations. 4. Preparations maintained in 1.1 mM calcium were used to compare inotropic responses to isoprenaline and tolbutamide at different frequencies of stimulation. 5. Decreasing the frequency of stimulation from 4 to 2 or 1 Hz decreased the resting force of contraction and increased the maximal inotropic responses to isoprenaline. There was an associated increase in EC50 values. 6. No significant inotropic responses to tolbutamide were observed at 4 Hz. Positive inotropic responses of similar magnitude occurred at 2 and 1 Hz. The maximal inotropic responses at these frequencies were 43- and 127-fold less than those to isoprenaline. 7. It is concluded that tolbutamide is a very weak inotropic agent.

Comparison of the beta-adrenoceptors in the myocardium and coronary vasculature of the kitten heart.

The relative potencies of (minus) noradrenaline, (minus)-adrenaline, (plus or minus)-isoprenaline and (plus or minus)-salbutamol have been assessed for their positive inotropic and chronotropic actions on kitten isolated atria. These relative potencies have been compared with those obtained for the relative coronary dilator potencies in two preparations. These were intact hearts perfused by Langendorff's method and isolated perfused coronary arteries from the kitten. The relative molar potencies for inotropic effects were noradrenaline 1: adrenaline1: iso-prenaline 7: salbutamol 0.6. The observed ratios for chronotropic effects were not significantly different from those for inotropic effects. The relative potencies of noradrenaline, adrenaline and isoprenaline as myocardial stimulants were similar to their relative potencies as coronary dilators in the intact heart. Similar relative potencies for noradrenaline and isoprenaline were also found in the isolated coronary artery but adrenaline was only one third as active as noradrenaline on this preparation. In both the intact heart and in the isolated coronary artery salbutamol was about one tenth as active as noradrenaline. It was therefore less active relative to noradrenaline as a coronary dilator than as a myocardial stimulant. In spite of these differences in relative potency ratios for myocardial and coronary dilator effects, similar dissociation constants (Kb values) for practolol against isoprenaline were found in driven atria and in isolated coronary arteries. Myocardial and coronary vascular beta-adrenoceptors thus can both be placed in the general classification of beta1-adrenoceptors.