Bernard Dufy

Potassium conductance in the androgen-sensitive prostate cancer cell line, LNCaP : Involvement in cell proliferation

Very little is known about the expression of ion channels in prostate cells (both normal and malignant), and their possible role in physiological and pathological functions. We therefore studied ion conductances and their role in the proliferation of LNCaP cells, an androgen‐sensitive human prostate cancer cell line.

Stimulation of Ca2+ influx in rat pituitary cells under exposure to a 50 Hz magnetic field

The effect of exposure of single rat pituitary cells to 50 Hz sine wave magnetic fields of various strengths on the intracellular free Ca2+ concentration ([Ca2+]i), was studied by using dual-emission microfluorimetry, using indo-1 as probe. A 30 min exposure of the cells to vertical 50 microT peak magnetic field triggered a long-lasting increase in [Ca2+]i from a basal value of about 185 +/- 4 nM to 326 +/- 41 nM (S.E.; n = 150). The vertical and horizontal components of the static magnetic field were 57 and 15 microT, respectively. The 50 Hz ambient magnetic field was always below 0.1 microT rms. The effect was observed both at 25 +/- 2 degrees C and at 37 +/- 2 degrees C. Responsive cells, for which [Ca2+]i rose to values above 309 nM, were identified as lactotrophs and represented 29% of the total pituitaries. [Ca2+]i increase, for the most part, was due to Ca2+ influx through voltage-dependent dihydropiridine-sensitive calcium channels inhibited by PN 200-110. However, neither Ca2+ channel blockers nor removal of Ca2+ from the external medium during exposure completely prevented the field-induced [Ca2+]i increase. Additional experiments using an MTT colorimetric assay showed that alteration of Ca2+ homeostasis of lactotrophs was associated with impairment of some mitochondrial processes.

Role of tyrosine phosphorylation in potassium channel activation. Functional association with prolactin receptor and JAK2 tyrosine kinase.

Chinese hamster ovary (CHO) cells, stably transfected with the long form of the prolactin (PRL) receptor (PRL-R) cDNA, were used for PRL-R signal transduction studies. Patch-clamp technique in whole cell and cell-free configurations were employed. Exposure of transfected CHO cells to 5 nM PRL led to the increase of Ca2+- and voltage-dependent K+ channel (KCa) activity. The effect was direct as it was observed also in excised patch experiments. A series of tyrosine kinase inhibitors was studied to investigate the possible involvement of protein tyrosine kinases in KCa functioning and its stimulation by PRL. Genistein, lavendustin A, and herbimycin A decreased in a concentration and time-dependent manner the amplitude of the KCa current in whole cell and the open probability of KCa channels in cell-free experiments. The subsequent application of PRL was ineffective. The protein tyrosine phosphatase inhibitor orthovanadate (1 mM) stimulated KCa channel activity in excised patches, indicating that channels can be modulated in opposite directions by protein tyrosine kinase and protein tyrosine phosphatase. Moreover, in whole cell experiments as well as in excised patch recordings, anti-JAK2 tyrosine kinase antibody decreased the KCa conductance and the open probability of the KCa channels. Subsequent application of PRL was no longer able to stimulate KCa conductance. Immunoblotting studies using the same anti-JAK2 antibody, revealed the constitutive association of JAK2 kinase with PRL-R. Preincubation of anti-JAK2 antibody with the JAK2 Immunizing Peptide abolished the effects observed using anti-JAK2 antibody alone in both electrophysiological and immunoblotting studies. We conclude from these findings that these KCa channels are regulated through tyrosine phosphorylation/dephosphorylation; JAK2 tyrosine kinase, constitutively associated with PRL-R, is implicated in PRL stimulation of KCa channels.

Measurement of Ca2+ transients using simultaneous dual-emission microspectrofluorimetry and electrophysiology in individual pituitary cells

Cytosolic free calcium concentration, [Ca2+]i, was monitored in individual pituitary GH3B6 cells, loaded with the fluorescent Ca2+ indicator indo 1 either by internal perfusion through a patch clamp pipette or by exposure to indo 1 acetoxymethyl ester, with the use of a dual-emission apparatus for microspectrofluorimetry. This system was sensitive enough to allow on-line monitoring of [Ca2+]i (from the ratio of fluorescent intensities) which could be combined simultaneously with whole-cell patch clamp recordings. The following situations were examined: (a) [Ca2+]i oscillations due to action potential firing, and (b) rapid transient elevations of [Ca2+]i triggered by voltage-dependent Ca2+ current. The results indicate that monitoring of [Ca2+]i at the single cell level with indo 1 provides a powerful means to study the [Ca2+]i regulation in pituitary cells, which should be applicable to many other cell types.

Effects of estrogen on the electrical activity of identified and unidentified hypothalamic units.

Experiments performed on unanesthetized ovariectomized female rabbits demonstrated the effects of estradiol benzoate (EB; 20 microng i.v.) on the electrical activity of hypothalamic units which send their axons to the median eminence. Of a total of 1,840 cells recorded in hypothalamic and preoptic areas, 46 (2.5%) were antidromically activated by stimulating the median eminence. Under the present experimental conditions, EB induced a progressive diminution in the mean firing rate of these cells observed throughout the recording period (30-120 min). In addition to cells projecting to the median eminence, neurons which could not be antidromically invaded using our techniques were observed to be sensitive to estrogen. Estrogen administration produced a long-lasting inhibition of antidromically activated cells and a depression of much shorter duration (15-20 min) of unidentified nonstimulated units. These data suggest the existence of two types of hypothalamic neurons sensitive to estrogen.

Rundown of GH3 cell K+ conductance response to TRH following patch recording can be obviated with GH3 cell extract.

GH3/B6 pituitary cells release prolactin (PRL) in response to thyrotropin releasing hormone (TRH). Electrophysiological assays of individual GH3 cells with sharp high-resistance microelectrodes have revealed complex effects of TRH on membrane excitability consisting of a transient hyperpolarization (1), which is thought to result from activation of Ca-dependent K+ conductance (2), followed by a prolonged phase of spontaneous, Ca-dependent action potential activity (3). Using the whole-cell patch recording (WCR) technique (4), we have found that these TRH actions on GH3 excitability rapidly rundown following patch recording. When the supernatant from osmotically lysed GH3 cells was added to the WCR patch pipette, the K+ conductance response was not only promoted but well-maintained. The results indicate that diffusible factors mediate these TRH actions and further, that the WCR technique should be useful in identifying different second messengers and elucidating their roles in membrane excitability and PRL secretion.

Short-term effect of estrogen on release of prolactin by pituitary cells in culture

Summary Whereas the long-term effect of estrogen on prolactin (PRL) secretion by the pituitary is now well known, the present study demonstrates a short-term stimulatory effect of 17 β-Estradiol (17 β-E) on PRL release from cloned pituitary cells (GH3/B6). The time-course of PRL release in the presence of the steroid showed a maximal increase of 86% over the control level after 10 min incubation. This effect was dose dependent and was not mimicked by the 17 α stereoisomer.

Bradykinin Parallels Thyrotropin-Releasing Hormone Actions on Prolactin Release from Rat Anterior Pituitary Cells

Bradykinin (BK), a nonapeptide, originally discovered in blood, is also present in neurons and fibers of the hypothalamus. We tested the putative releasing factor properties of BK on prolactin (PRL) release from anterior pituitary cells in vitro. BK stimulated the release of PRL in a dose-dependent manner, the threshold concentration being in the range. 0.1-1.0 nM. The release of PRL induced by BK at 1 nM concentration was about 2-fold, delayed and sustained over many minutes. Higher concentrations of BK stimulated PRL release in two phases. The shape of the BK-induced PRL release was superficially similar to that induced by thyrotropin-releasing hormone (TRH). 10 nM BK and 10 nM TRH induced about a 4-fold increase in PRL release within 5 min, followed by a gradual recovery to basal secretion. These results indicate that this peptide can act directly at the anterior pituitary gland to release PRL. Phorbol ester also promoted PRL release over the range of 1-10 nM, but the time course of the release was somewhat different.

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

Short Applications of Gamma-Aminobutyric Acid Increase Intracellular Calcium Concentrations in Single Identified Rat Lactotrophs

We have investigated the direct effect of GABA receptor agonists on the cytosolic free calcium concentration ([Ca2+]i) and the membrane potential of rat lactotrophs in primary culture. [Ca2+]i was recorded in single identified lactotrophs by dual emission microspectrofluorimetry using indo-1 as intracellular fluorescent calcium probe. Whole cell and perforated patch-clamp were performed. A short application of GABA (10(-5) M, 10 s) induced a marked transient [Ca2+]i increase in 66% of lactotrophs, which could be readily mimicked by muscimol (10(-5) M). By contrast, neither L-homocarnosine (10(-3) M) nor baclofen (10(-5) M), a GABAB agonist, had any effect on [Ca2+]i. The GABA-induced [Ca2+]i increase was antagonized by picrotoxin (10(-5) M), bicuculline methiodide (10(-5) M) and strychnine (10(-4) M), demonstrating GABAA receptor specificity. Furthermore, clonazepan (1.5 x 10(-4) M) could potentiate the GABA effect on [Ca2+]i. The [Ca2+]i increase disappeared in the absence of Ca2+ in the extracellular medium or in the presence of Ca2+ channel blockers (cadmium, PN 200-110). GABA and muscimol depolarized the membrane potential with a concomitant fall in cell input resistance, thus suggesting, as in other cell types, the opening of receptor-operated chloride channels. When Ca2+ entry was prevented by the use of cadmium (500 x 10(-6) M), GABA still elicited membrane depolarization but did not raise [Ca2+]i. Our results suggest that a short application of GABA leads to Ca2+ entry through voltage-gated Ca2+ channels in single lactotrophs. This Ca2+ influx is due to depolarization of the prolactin cell.