17 beta-estradiol stimulates a rapid Ca2+ influx in LNCaP human prostate cancer cells.

Prostate growth is known to be controlled by steroids such as androgens and estradiol. For this reason steroids (estradiol, adrenal androgens) or steroid inhibitors are commonly used as palliative treatments for prostate carcinoma. In view of the pivotal role played by Ca2+ ions in cell proliferation, we decided to investigate the effects of 17 beta-estradiol (E2) on intracellular calcium concentration ([Ca2+]i) in a human prostate tumor cell line. LNCaP. In this study, we show that E2 induced a dose-dependent (0.1-100 nmol/l) influx of Ca2+ in these cells. These effects occurred rap dly after the beginning of the ejection and were maintained in the presence of the hormone (plateau phase). Estradiol-induced Ca2+ influx was unaffected by the saturation of the androgen receptor with pure antiandrogen flutamide. The use of tamoxifen, an antiestrogen binding to nuclear receptors, or E2 covalently linked to bovine serum albumin that cannot penetrate the cell membrane, did not block the ([Ca2+]i) response. Our results suggest the existence of E2 binding sites at the plasma membrane surface of LNCaP cells, linked to calcium signalling and, more specifically, Ca2+ channels.

Dual Effect of Prolactin on Protein Kinase C Activity in CHO Cells Expressing Functional Prolactin Receptors.

We investigated the effects of prolactin (PRL) on the protein kinase C (PKC) activity in Chinese hamster ovary (CHO-E32) cells stably transfected with rabbit mammary gland PRL receptor cDNA. These cells express a functional long form of PRL-R. A 10-min to 2-hour treatment with 5 nM PRL resulted in the translocation of PKC activity from the cytosol to the membrane. Longer treatment (10-24 h) with the same concentration of PRL decreased the PKC activity in both particulate and cytoplasmic fractions. The PRL effect was dose dependent: maximal action was obtained with 1-10 nM. The PRL-induced activation of PKC was blocked by 20 nM staurosporine, a PKC inhibitor. Two inhibitors of tyrosine kinase, herbimycin A (1.75 &mgr;M) and genistein (100 &mgr;M), had no effect on PRL-induced activation of PKC. Copyright 1996 S. Karger AG, Basel

The Lipidosterolic Extract fromSerenoa repens Interferes with Prolactin Receptor Signal Transduction.

The lipidosterolic extract from the saw palmetto Serenoa repens (LSESr) is commonly used for medical treatment of benign prostatic hypertrophia due to its ability to inhibit 5alpha-reductase which permits the conversion of testosterone to dihydrotestosterone, the active androgen on prostate cell proliferation. However, the complete action mechanism of LSESr is still unknown. Several lines of evidence suggest that, in addition to inhibition of 5alpha-reductase, it may interfere with the action of prolactin (PRL). We therefore investigated a possible interference of this plant extract with another hormone that controls prostate gland growth, PRL. As the action mechanism of PRL is now fully documented in Chinese hamster ovary cells expressing the PRL receptor, we have conducted our experiments on these cells. In this study, using electrophysiological (whole-cell recording and single-channel recording), microspectrofluorimetric and biochemical techniques, we show that LSESr (1-30 &mgr;g/ml) reduced the basal activity of a K(+) channel and of protein kinase C (PKC) in CHO cells. In addition, pretreatment of the cells with 1-10 &mgr;g/ml LSESr for 6-36 h abolished the effects of PRL on [Ca(2+)](i), K(+) conductance and PKC. LSESr may block PRL-induced prostate growth by inhibiting several steps of PRL receptor signal transduction. LSESr may also be useful for diseases implicating PRL. Copyright 1995 S. Karger AG, Basel

Estradiol modulation of GNRH-stimulated LH release in rat anterior pituitary cells: involvement of dihydropyridine-sensitive calcium channels.

Although enhancement of GnRH-stimulated luteinizing hormone (LH) release by estradiol (E2) has been established, it is not known at what stages of the process of transduction E2 acts. We investigated the release of LH in response to GnRH and to Bay K 8644, an activator of L-type calcium channels, in a culture of pituitary cells obtained from ovariectomized females, these cells having being treated or not with E2 (OVX + E2 and OVX). We studied the effects of D600, an antagonist of T- and L-type calcium channels, and PN 200-110, an antagonist of L-type calcium channels. The effects of the latter were studied in protein kinase C-depleted cells in order to investigate the possible phosphorylation of these channels. D600 caused a decrease in GnRH-stimulated LH release in OVX and OVX + E2 cells. However, this decrease was greater in OVX + E2 cells, suggesting that at least one type of calcium channels may be involved as a result of treatment with E2. We confirmed the involvement of L-type calcium channels in the action of GnRH since the GnRH-stimulated LH release was enhanced in the presence of Bay K 8644 in OVX cells. Bay K 8644 alone increased basal LH in a dose-dependent manner only in OVX + E2 cells. PN 200-110 induced a decrease of GnRH-stimulated LH release only in OVX + E2 cells. These results suggest that L-type calcium channels are activated in E2-treated cells. The dose-dependent decrease caused by PN 200-110 in OVX + E2 cells disappeared in the OVX + E2 PKC-depleted cells. This result was confirmed with Bay K 8644 and suggests a phosphorylation of dihydropyridine-sensitive calcium channels by protein kinase C.

Biphasic changes in intracellular pH induced by thyrotropin-releasing hormone in pituitary cells.

We studied the effects of TRH on intracellular pH (pHi) in individual cells of the GH3 pituitary clonal cell line using the seminaphtorhodafluor pH indicator. We show that, in a majority of cells, TRH action on pHi occurs in two phases: first acidification then alkalinization. Acidification and Ca2+ mobilization are related in time. K+ depolarization (KCl, 50 mM), and Ca2+ ionophores, A23187 (10 microM) or ionomycin (5 microM) lead to acidification. We conclude that a marked increase in [Ca2+]i can induce acidification and that the TRH-induced acidification is due to Ca2+ mobilization. TRH-induced alkalinization is due to Na+/H+ exchanger activation, since it is inhibited by amiloride (200 microM) and Na(+)-free medium. We show that this alkalinization does not occur after a 20-h pretreatment with phorbol myristate acetate (1 microM) which depletes protein kinase C. We also show that blocking Ca2+ entry does not affect the TRH-induced alkalinization, but an increase in [Ca2+]i concomitant with the activation of protein kinase C mimics TRH-induced alkalinization. We conclude that both Ca2+ mobilization and protein kinase C activation are necessary for TRH-induced alkalinization. Studies of secretion in Na(+)-free medium or with amiloride (200 microM) show that pHi does not seem to be involved in PRL short-term release (30 min) but suggest that activation of the Na+/H+ exchanger leading to cytoplasmic alkalinization may have an important role in PRL synthesis.

Gonadotropin-releasing hormone-induced changes of intracellular pH in pituitary gonadotrophs: influence of estradiol.

Using the pH indicator, seminaphtorhodafluor, we studied the effects of GnRH on intracellular pH (pHi) in single gonadotroph cells, obtained from 3-week ovariectomized rats, treated or not with estradiol (E2) (OVX + E2, OVX). In a majority of cells (77.7% for OVX cells and 93.7% for OVX + E2 cells), GnRH induced acidification. A biphasic change of pHi, acidification followed by alkalinization, was observed in about 44% of the cells tested. In E2-treated cells, amplitude of acidification and duration of alkalinization were increased. Acidification and Ca2+ mobilization were related in time with a short delay (4-5 sec.). Depolarization with KCl and ionomycin, a Ca2+ ionophore, induced acidification. Taken together these observations suggest that acidification was caused by [Ca2+]i increase. When the Na+/H+ exchanger was blocked by amiloride or in Na(+)-free medium, GnRH-induced alkalinization was inhibited. Alkalinization disappeared completely when the cells were depleted in protein kinase C (PKC). Nevertheless, acute application of phorbol myristate acetate, known to activate PKC, was not sufficient to induce alkalinization. We conclude that PKC is necessary but not sufficient for alkalinization. In contrast, the GnRH response can be mimicked by a simultaneous application of phorbol myristate acetate and KCl. To further explore the putative role of pHi in the secretory process, LH release was studied. Using Na(+)-free medium or amiloride, we show that basal LH was not dependent upon the Na+/H+ exchanger activity. Conversely, GnRH-induced LH release was significantly decreased; this decrease was greater in E2-treated cells but prevented by bicarbonate. These data show that pHi and the Na+/H+ exchanger play an important role in the stimulus secretion coupling process of gonadotrophs. E2, which is an important factor in the regulation of gonadotropic hormone release, participates also in the pHi variations.

Transient but not oscillating component of the calcium mobilizing response to gonadotropin-releasing hormone depends on calcium influx in pituitary gonadotrophs.

Gonadotropin-releasing hormone (GnRH)-stimulated changes in the cytosolic free Ca2+ concentration ([Ca2+]i) were studied in gonadotrophs cultured from 3-week ovariectomized rat pituitaries. One animal was used per cell preparation. [Ca2+]i was monitored in individual gonadotrophs by dual emission microspectrofluorimetry, using Indo-1 as the intracellular fluorescent Ca2+ probe. A short stimulation with GnRH evoked a complex concentration-dependent Ca2+ response in individual gonadotrophs. 0.1-1 nM GnRH triggered a series of sinusoidal-like [Ca2+]i oscillations superimposed upon a modest slow [Ca2+]i rise--the oscillating response mode--while 10-100 nM GnRH caused a biphasic increase in [Ca2+]i consisting of a monophasic transient and oscillations--the transient/oscillating response mode. Despite the consistency of Ca2+ responses, an inter-preparation heterogeneity of [Ca2+]i oscillations frequency was noticed. Moreover, we observed that, within a given cell preparation, the frequency of [Ca2+]i oscillations was independent of GnRH concentration whereas both peak [Ca2+]i and area under the [Ca2+]i versus time curve were concentration-dependent. Thus, in gonadotrophs, the presence of the GnRH signal would lead to [Ca2+]i oscillations, while the amplitude of the [Ca2+]i responses would code for the concentration of agonist. Both transient and oscillating components of GnRH responses depended on releasing activity of Ca(2+)-sequestering pools in as much as GnRH responses were unaffected by brief removal of external Ca2+, but suppressed by chelating intracellular free Ca2+ with BAPTA. However, prolonged exposure to a Ca(2+)-free medium suppressed the transient component while leaving the oscillating component unaffected. We therefore propose that gonadotrophs employ Ca(2+)-sequestering pools, whose maintenance depends on a slow Ca(2+)-entry, to give an amplitude-coded Ca2+ rise in response to a short GnRH stimulation.

Estrogen modulated gonadotropin release in relation to gonadotropin-releasing-hormone (GnRH) and phorbol ester (PMA) actions in superfused rat pituitary cells.

The involvement of protein kinase C (PKC) in GnRH action is still a matter of controversy. We have conducted a comparative study of LH and FSH release in response to GnRH and to phorbol ester myristate acetate (PMA), an activator of PKC, by rat pituitary cells maintained in culture. The effect of E2 pretreatment coupled or not with PKC depletion was also studied. Different kinetics in the response of LH and FSH to GnRH were observed, suggesting that the intracellular pathways involved in the release process of the two hormones were somewhat different. Moreover, PMA (10 nM) stimulated LH release greatly and FSH release only slightly. Intracellular PKC depletion, obtained by a prolonged treatment (18 h) of the cells with PMA (1 microM), produced different results according to the endocrine status of the pituitary cells. GnRH (10 nM)-induced LH release was significantly decreased in PKC-depleted cells from proestrous females. For PKC-depleted cells from OVX females, it was decreased significantly only when cells had been pretreated by E2. These results suggest that the modulation of LH secretion by E2 involves PKC activation. FSH release was poorly stimulated by PMA; but, under any conditions, PKC depletion did not affect GnRH-induced FSH release.

[Modulation of LH release by estradiol. Involvement of protein kinase C in the action mechanism of GnRH]. / Modulation par l'oestradiol de la libération de LH. Implication de la protéine kinase C dans le mécanisme d'action du GnRH.

The involvement of PKC in GnRH action is still controversial. Discrepancies between different results could be due to the endocrine status of cells used for the studies. In order to determine a putative role for PKC in GnRH action and if gonadal steroids could be implicated in the PKC contribution to GnRH action, we have conducted a study of LH release in response to GnRH and to PMA, an activator of PKC, using an anterior pituitary cell culture system. The direct effects of E2 were considered coupled or not with the effect of PKC depletion. GnRH and PMA induced LH releases in a dose-dependent manner. Both are increased by E2. The PKC depletion had no effect on GnRH stimulated LH release in cells deprived of gonadal steroid influence but induced a significant decrease in cells which had been treated by E2. These results indicate that E2 alters cell sensitivity to GnRH by affecting post-receptor intracellular pathways such as PKC activation.

[LHRH and LH release in the red fox Vulpes vulpes L]. / LHRH et libération de LH chez le Renard roux Vulpes vulpes L.

Pituitary responsiveness to exogenous LHRH was studied in vivo and in vitro in the female red fox, a mono-oestrous species. In vivo, the ability of the pituitary to release LH in response to a single injection of LHRH (2 micrograms/kg) was determined at various stages of the reproductive cycle. The greatest responsiveness is observed during the preovulatory period, the lowest during the luteal phase. During the anoestrus phase, the responsiveness is reduced by more than 50% in lactating females compared to non lactating females. In vitro, dispersed fox anterior pituitary cells were exposed four times to LHRH (10(-9) M), hourly, for 8 min. Pituitary cells were taken from lactating and non lactating females. The cells are not sensitive to LHRH in lactating females but become more and more sensitive after weaning. It is suggested the inhibitory influence of lactation could be the result of prolactin-ovarian steroids-gonadotrophins interactions.

Seasonal variations in plasma luteinizing hormone and testosterone levels in the European badger Meles meles L.

Although active spermatogenesis occurs throughout the year in the male European badger Meles meles L., seasonal fluctuations of testicular activity were observed. The present study compared the pattern of luteinizing hormone (LH) and testosterone (T) secretions throughout the annual cycle. LH secretion was investigated by means of a heterologous radioimmunoassay using anti-ovine LH beta antiserum and canine reference standard. Seasonal and nycthemeral changes in LH and T secretions were observed. During the year a period of maximal values for both hormones is well correlated with the breeding. Moreover, episodic LH releases occurs during the period of minimal testosterone values, indicating resumption of hypothalamo-pituitary activity well before the breeding period.

C21 steroids and transcortin-type protein during delayed implantation in the European badger Meles meles L.

A high-affinity corticosteroid-binding protein (CBG) roughly resembling a transcortin-type protein is present in badger plasma. Plasma CBG, corticosteroid and progesterone (P) concentrations were measured in relation to delayed implantation, true progestation and gestation. Two significant CBG increases were observed during pregnancy. The first, in the second half of embryonic diapause coincides with an increase in plasma corticosteroid concentration and the second, during true progestation and gestation, with an increase in P concentration. Relationship of CBG increases with pregnancy in badger are discussed.

[Seasonal variations in the nyctohemeral rhythm of plasma testosterone in the European badger Meles meles L]. / Variations saisonnières du rythme nycthemeral de la testostérone plasmatique chez le blaireau européen Meles meles L.

In the badger a seasonal sexual rhythm of the plasma testosterone is observed. There is also a nycthemeral rhythm of the testosterone secretion; during the regressed period of the testis this rhythm is bimodal with testosterone peaks during the light phase of the day; during the active period and "activity rhythm" with many transitorys peaks during the dark phase is added.

[Seasonal variations in the sexual activity of the male genet (Genetta genetta L.)]. / Variations saisonnières de l'activité sexuelle de la genette mâle (Genetta genetta L.).

The Genet testis shows a spermatogenic activity throughout the year. The plasmatic androgen level presents two periods where the values are higher; one in spring the other in autumn. The spring testosterone rise coïncides with a mating period.

[Seasonal plasma testosterone variations in the stone marten]. / Variations saisonnières de la testostérone plasmatique chez la fouine.

Plasma testosterone levels change with annual sexual cycle of the stone marten. Very low during the decrease and the quiescence of the testis, it rises during the testis onset and reaches a maximal peak during the coït period (May to July).

[Photoperiod effect testicular physiology in the stone-marten (Martes foina Erx.)]. / Influence de photopériodisme sur la physiologie testiculaire de la fouine (Martes foina Erx.)

The male Stone-Marten (Martes foina) has a well-defined sexual cycle with two very different phases: a breeding season (May-June) and a quiescent period (November-December). We can cause the onset of the testicular activity, during the quiescent phase, by an experimental increase of daylight (18 L-6 D).