stimulated rates of release of lutropin. By contrast, flufenamic acid and meclofenamic acid speciJically inhibited the stimulation of cyclic AMP by luliberin.
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reaction with prostaglandin F. Lutropin was measured by homologous radioimmunoassay by using NIAMD-LH-RP-1 as standard. It has been shown that the addition of prostaglandin precursors to anterior pituitaries in vitro leads to rapid increases in prostaglandin synthesis and increased accumulation of cyclic AMP (Bergeron & Barden, 1975). However, a concentration of indomethacin (lOpg/ml), which was effective at preventing this effect, failed to prevent the stimulation of cyclic AMP accumulation by luliberin (10mM) during a 3h incubation. Concentrations of indomethacin of 10,ug/ml or more did not inhibit either the basal or luliberinstimulated rates of release of lutropin. By contrast, flufenamic acid and meclofenamic acid speciJically inhibited the stimulation of cyclic AMP by luliberin. Doses of 50pg of meclofenamate/ml or 1OOpg of flufenamate/ml caused complete inhibition. Concentrations of fenamates of up to 50,uglrnl also inhibited luliberin-dependent lutropin release, whereas higher concentrations caused an increase in the rate of lutropin secretion, in the absence or presence of luliberin. It is possible that the fenamates may interact with other enzyme systems with the result that their effects on cyclic AMP concentrations and lutropin release may be independent of their effects on prostaglandin synthesis. The accumulation of prostaglandin E by dispersed pituitary cell cultures was increased fourfold by the addition of luliberin (IOnM), the maximum effect being observed after 2.5h of incubation (Fig. 1). The addition of indomethacin (50,uglrnl) or flufenamate (25pg/ml) completely abolished the effect of luliberin, lowering the intracellular concentration of prostaglandin E to below the control. The effect of indomethacin suggests that the stimulation of prostaglandin concentrations is not an essential part of the mechanism of action of luliberin on lutropin release. It is noteworthy that the effects of luliberin on cyclic AMP and prostaglandin accumulation occur at approximately the same time. It was found that hemipituitaries incubated with 2m~-N~-monobutyryl cyclic AMP, and dispersed cells incubated with 1m~-S-bromocyclic AMP or 2m~-theophylline,all responded with increased intracellular prostaglandin E accumulation. It is therefore possible that the effect of luliberin on prostaglandin concentrations is a consequence of its effect on cyclic AMP accumulation. Bergeron, L. & Barden, N. (1975) Mol. Cell. Endocrinol. 2,253-260 Borgeat, P., Labrie, F. & Garneau, P. (1975) Can.J. Biochern. 53,455-460 Chobsieng, P., Naor, Z., Koch, Y., Zor, U. & Lindler, H. (1975) Neuroendocrinology 17,12-I7 Gilman, A. (1970) Proc. Natl. Acad. Sci. U S A . 67, 305-312 Harms, P., Ojeda, S. & McCann, S. (1974) Endocrinology 94, 1459-1464 Labrie, F., Pelletier, G., Lemay, A., Borgeat, P., Barden, N., Dupont, A., Savary, M., 0 3 6 , J. & Boucher, R. (1973) Karolinska Symp. Res. Methods Reprod. Endocrinol. 6th, pp. 301-334 Sato, T., Hirono, M., Jyujo, T., Iesaka, T., Taya, T. & Igarashi, M. (1975) Endocrinology 96, 45-49
Pituitary Involvement in the Control of Hepatic Steroid Metabolism in the Rat PAUL SKETT, JAN-AKE GUSTAFSSON, AKE STENBERG Department of Chemistry I, Karolinska Institutet, S-104 01 Stockholm 60,Sweden
and AGNE LARSSON Department of Pediatrics, St. Gorans Hospital, S-112 81 Stockholm, Sweden
Sexual differences in hepatic steroid metabolism have been well characterized (Forchielli & Dorfman, 1956; Yates et al., 1958; Gustafsson & Stenberg, 1973a,b), but their control has been a subject of wide discussion. Experiments by Denef (1974), as well as by our own group (Gustafsson & Stenberg, 1974), indicated that the pituitary gland plays a significant role in the maintenance of the female pattern of metabolism and that the release of the pituitary factor involved was controlled in a similar manner to Vol. 4
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prolactin (i.e. predominantly by means of areleaseinhibiting factor from the hypothalamus). It is possible that the effects of androgens and oestrogens on hepatic steroid metabolism are indirect effects involving changes in the release of the pituitary factor(s) concerned. In order to investigate the nature of the pituitary factor(s) mentioned above, an assay system was developed using the steroid-metabolizingcapacity of a hepatoma cell line in culture (HTC cells). This cell line was originally isolated from a chemically induced hepatoma (no. 72884 produced in a rat of the Buffalo strain and has been shown to carry at least one liver-specific marker enzyme, a glucocorticoid-inducible tyrosine aminotransferase (Thompson et al., 1966). The cells were grown in minimum essential medium (with Earle's salts) supplemented with 5 % calf serum, 5 % foetal calf serum, penicillin (200i.u./ml) and streptomycin (125,ug/ml) in 60mm Petri dishes. Each dish, containing5ml of medium, was seeded with 5 x lo4 cells. After 4 days the medium was changed and the extracts to be tested were added. The cells were maintained for a further 3 days in the new medium and then harvested, washed free of medium with phosphate-bufferedsaline (pH7.4) and immediately analysed for enzyme activity, The method for determining the activities of the steroidmetabolizing enzymes has been described previously (Gustafsson et al., 1975) and uses 4-androstene-3,17-dione as substrate. Duplicate incubations were performed in all cases, and the results are expressed as pmol of product formed/min per mg of protein. Protein was determined by the method of Lowry et al. (1951), and statistical analyses were by Student's t test (the level of significance was set at 0.05). The major enzyme activity in the HTC cells was found t o be the Sa-reductase which, in the intact rat liver, is a sex-dependent enzyme (Einarsson et al., 1973). The high-molecular-weight fraction of homogenized female pituitary glands (separated on SephadexG-25) was shown to increase the Sa-reductaseactivity of HTC-cellhomogenates at subsaturation concentrations of the substrate, whereas the corresponding male extract was without effect. The response of the cells was linear with respect to log dose of pituitary extract over a wide range of concentrations of female pituitary extract. The change in the activity of the Sa-reductasewas thus taken as a measure of the activity of the pituitary factor(s) involved, in subsequent experiments. Studies on the kinetics of the 5areductase enzyme, before and after addition of female pituitary extract, showed that the apparent increase in the activity of the enzyme at subsaturation concentrations of substrate was due to a decrease in the apparent K,,, and not to an increase in apparent V,,,,,. (see Fig. 1). This is similar to the differences observed between the Sa-reductases in
lo-'/[s](K') Fig. 1. Efect of the concentration of 4-androstene-3,17-dioneon the Sa-reductase activity observed in homogenates of HTC cells with (0)and without (0)pretreatment withfemale pituitary extract in the culture medium for 3 days expressed as a double-reciprocal Lineweaver-Burk plot 1976
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- 10
- 8
- 6
-
%
- 4
- 2
0
0
I 2 3 4 5 6 7 8 910111213141516171819
Fraction no. Fig. 2. Distribution of yeminizing factor’ activity on an Ampholine column (pH3-10) stabilized with sucrose
-,
pH gradient in the column. Fractions of volume 1ml were collected.
gonadectomized male and female rats (the ‘increased‘ Sa-reductase in the female being due to a lower K,,,). A series of purified pituitary hormones were tested for activity in this assay (including lutropin, follitropin, prolactin, growth hormone, thyrotropin, corticotropin, oxytocin and vasopressin), but only follitropin showed any activity and this was only 15 % of’the activity of the female pituitary extract (based on the concentration of follitropin in fresh pituitary tissue). Isoelectric focusing of female pituitary extracts (see Fig. 2) indicated that the factor affecting Sa-reductase activity (to which we have given the name the ‘feminizing factor’ as it ‘feminizes’ the S/.-reductase activity) is a single protein with a PI of 8.3. This value further distinguishes it from the other pituitary hormones which, in general, have a PI below 8.0 (except lutropin, which in our experiments had a PI of 9.0). Separation of the secretory granules of the female pituitary gland by sucrose-gradient centrifugation (60min at 149000g continuous gradient, d = 1.11-1.26) showed that the feminizing factor is stored in granules with a density of 1.14g/cm3, again separating it from lutropin and follitropin, which are stored in much denser granules. The distribution pattern of the feminizing-factor granules was similar to that of prolactin granules. This work indicates that there is a factor in the pituitary that can influence Sa-reductase activity in HTC cells. The similarities between this effect and the changes in Sa-reductase in the intact liver suggests that this factor may also be the effector in the intact rat and be responsible for the altered Sa-reductase activity seen in the female when compared with the male animal. This factor is a protein with a PI of 8.3 and is stored in low-density granules in the pituitary gland. This work was supported by grants from the Swedish Medical Research Council (no. 03X2819) and from WHO. P. S.is grateful to the CIBA-GEIGY Fellowship Trust for a fellowship. Denef, C. (1974) Endoerimlogy 94,1577-1582 Einarsson, K . , Gustafsson, .J.-A. & Stenberg, A. (1973) J. Biol. Chem. 248,4987-4997 Forchielli, E. & Dorfman, R.I. (1956) J. Biol. Chem. 223, 443-448 Gustafsson, J.-A. & Stenberg, A. (1973~)J. Biol. Chem. 249,711-718
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Gustafsson, J.-A. & Stenberg, A. (19736) J. Biol. Chem. 249,719-723 Gustafsson, J.-A. & Stenberg, A. (1974) Endocrinology 95, 891-896 Gustafsson, J.-A., Larsson, A., Skett, P. & Stenberg, A. (1975) Proc. Narl. Acad. Sci. U.S.A. 72, in the press Lowry, 0.H., Rosebrough, N. J., Farr, A. L. &Randall, R. J. (1951)J. Biol. Chem. 193,265-275 Thompson, E. B., Tomkins, G. M. & Curran, J. F. (1966) Proc. Natl. Acad. Sci. U.S.A. 56, 296-303
Yates, F. E., Herbst, A. L. & Urquhart, J. (1958) Endocrinology 63, 887-902
The Effect of Spermidine on the Uptake of Tryptophan and Corticosteroids by the Isolated Perfused Guinea-Pig Mammary Gland A. R. PETERS and T. B. MEPHAM Department of Physiology and Environmental Studies, University of Nottingham, Faculty of Agricultural Science, Sutton Bonington, Loughborough, Leics., U.K.
Studies have suggested an important role for the polyamine, spermidine, in lactogenesis. Russell & McVicker (1972) reported that the concentration of spermidine in rat mammary glands increases markedly during lactation, whereas Oka (1974) showed that it could to a large extent substitute for cortisol in the hormone triplet (cortisol, insulin and prolactin) that stimulates milk protein and eniyme synthesis in mouse mammary explants cultured in vitro. We report here the results of experiments on the possible role of spermidine in established lactation, by using the isolated perfused mammary-gland preparation described by Davis & Mepham (1974). This system permits the measurement of substrate and hormone uptake over a period of several hours, during which physiological perfusate flow rates and milk secretion are maintained. During the perfusions single-pulse injections of spermidine were given to a final perfusate concentration of about 5mmol/litre, i.e. the approximate concentration of spermidine in lactating rat mammary gland (Russell & McVicker, 1972). The mammary uptake of glucose, individual amino acids and corticosteroids were measured by sampling the perfusate at 20min intervals. Of the substrates measured, only tryptophan (assayed by the method of Denckla & Dewey, 1967) showed a significant response to spermidine administration. The uptake of tryptophan was markedly increased over the 20min period after spermidine injection, although it invariably fell over the subsequent 20min period to a value below the mean uptake before injection. The response was significantly correlated with perfusate tryptophan concentration in the range 5-150pg/ml (PcO.001), although there was no correlation between uptake and perfusate concentration in the absence of exogenous spermidine. Responses were not always observed when the tryptophan concentration was in the physiological range (i.e. 5-30pg/ml). Results from the ten experiments performed show that the mean tryptophan uptake was 1.13+0.15mg/h (mean? s.E.M.) over the period before, and 3.00+0.64mg/h over the 20min period after spermidine injection. However, owing to the decreased uptake in the ensuing 20min period, the mean uptake during the 40min period after spermidine treatment was 1.48mg/h. When a second spermidine injection was given 60min after the first, a marked stimulation of tryptophan uptake again resulted. The uptake of corticosteroids (assayed by a modification of the method of Bassett & Hinks, 1969), of which over 90% is believed to be in the form of cortisol (Illingworth er al., 1974), also showed amarked response to spermidine administration. In five experiments the mean uptake was 5.70f0.95pg/h over the period before treatment, -8.06pg/h during the 20min period after spermidine injection, and 5.00f 1.72pg/h during the subsequent 20min period. There was no correlation between the magnitude of the response and the perfusate corticosteroid concentration, which was in the range 85.7373.3 ng/ml in the five experiments. 1976
