Benoît P. Winiger

Delayed sexual maturation induced by daily melatonin administration eliminates the LH response to naloxone despite normal responsiveness to GnRH in juvenile male rats

Daily administration of melatonin (MT) markedly delays sexual maturation in the male Wistar rat. In this study, we have evaluated pituitary responsiveness to GnRH and the level of tonic inhibition by endogenous opioids in normal juvenile male rats and in rats with delayed sexual development induced by daily afternoon MT injection (100 micrograms, s.c.) starting at 20 days of life. Plasma LH responses to repetitive intravenous GnRH administration (100 ng/100 g body weight), or to different doses of GnRH administered subcutaneously (5-100 ng/100 g body weight) were normal in MT-treated rats both at 30 and 40 days of life despite significantly lower number of pituitary GnRH receptors and decreased pituitary gonadotropin content. One naloxone (NAL) injection (2.5-5.0 mg/kg, s.c.) produced a significant increase of plasma LH in normal 40- and 55-day-old rats, which was not seen in MT-treated rats of the same age. In contrast, no increase of plasma LH was seen in 30-day-old control rats nor in MT-treated rats at this age. Pretreatment with morphine sulfate (10 mg/kg, s.c.), or with the potent Met-enkephalin analog FK 33-824 (1.0 mg/kg, s.c.) prevented the NAL-induced rise of plasma LH in control rats at day 40 of life. In all instances, plasma PRL levels were decreased after NAL both in untreated and in MT-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatostatin inhibition of hormone release: Effects on cytosolic Ca++ and interference with distal secretory events

In normal pituitary cells somatostatin (SRIF) blocks the spontaneous oscillations in [Ca++]i by inhibiting the generation of action potentials. This is sufficient to explain the inhibitory effect on basal, but not entirely that on stimulated pituitary hormone secretion. In insulin secreting cells, which, in contrast to pituitary cells, only fire action potentials on stimulus-evoked depolarization, SRIF hyperpolarization and lowering of [Ca++]i is only transient. The marked inhibition of insulin secretion is suggested to be due to a coordinated action of SRIF on membrane potential and [Ca++]i as well as a direct interference with late secretory events.

Monitoring Receptor Mediated Regulation of Cytosolic Calcium in Single Pituitary Cells by Dual Excitation Microfluorimetry

Receptor-mediated alterations in the cytosolic free calcium concentration, [Ca2+]i are monitored with the intracellular fluorescent calcium probe fura 2 by dual excitation microfluorimetry. The calcium dependence on the excitation spectrum of fura 2 allows us to choose two wavelengths, lambda 1 and lambda 2, at which an increase in [Ca2+]i causes either a rise or a fall in fluorescence; the ratio of fluorescence at lambda 1 and lambda 2 (R = F lambda 1/F lambda 2) is a measure of [Ca2+]i. It appears essential for such measurements that the alteration between the two excitation wavelengths is done rapidly, to allow us to distinguish between effects on [Ca2+]i and other effects on fluorescence. In addition, specific problems relating to the calibration of fura 2 measurements, such as its relative insensitivity at basal [Ca2+]i, the role of intracellular viscosity on fura 2 fluorescence, and the difficulties encountered in establishing calibration constants have to be appreciated. In spite of these potential drawbacks, it appears that monitoring receptor-mediated [Ca2+]i regulation in single cells will prove essential for the further comprehension of stimulus-secretion coupling in pituitary and probably many other cell types.

Solubilization of pituitary GnRH binding sites by means of a zwitterionic detergent

Solubilization in an active form of the pituitary plasma membrane protein carrying binding sites for gonadotropin-releasing hormone (GnRH) was possible by using as detergent CHAPS (3[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate), which is a zwitterionic derivative of cholic acid. In contrast, the use of the non-ionic detergent Triton X-100 resulted in the loss of GnRH binding sites. CHAPS at a concentration of 5-10 mM allowed the solubilization of up to 80% of available sites, and remaining non-solubilized sites retained unaltered binding properties. Characterization of solubilized binding sites of GnRH was achieved by using as radioactive ligand and standard a stable GnRH analog. [D-Trp6,(NEt)Pro9,desGly10]-GnRH. Maximum binding of GnRH to solubilized binding sites was possible in the presence of a very low amount of CHAPS in the incubation medium. Scatchard analysis of classical saturation experiments indicated the presence of a single class of high affinity, low capacity binding sites. The mean value for the affinity constant KA was 0.36 +/- 0.07 X 10(10) M -1 (n = 5) for solubilized GnRH binding sites, when analysed in the presence of 2 mM CHAPS; when solubilized GnRH binding sites were analysed in the absence of CHAPS, significantly higher KA values were observed, ranging from 1.2 to 5.9 X 10(10) M -1. Parallel analysis of the plasma membranes prior to solubilization indicated a mean KA value of 0.53 +/- 0.13 X 10(10) M -1 (n = 5). The binding specificity of the pituitary GnRH binding sites as seen by evaluating cross-reactions with a panel of agonists and antagonists of GnRH was not altered by solubilization. Gel filtration on Sephadex G-25 of solubilized binding sites preincubated with radioiodinated GnRH analog demonstrated the presence of a large molecular weight complex. In conclusion, CHAPS appears to be quite suitable for the solubilization of the binding site moiety of the pituitary GnRH receptor with good retention of the binding characteristics, thus offering a new possibility for the purification and characterization of the GnRH receptor protein.

Inhibitors of 1,2-diacylglycerol kinase potentiate the TRH-induced stimulation of Ca2+-activated K+ current

Transient activation of the outward K+ current caused by a rise in the cytosolic free Ca2+ concentration, [Ca2+]i was the predominant change in plasma membrane ion flux during the first phase of thyrotropin-releasing hormone (TRH) action on pituitary cells. Following the intracellular application of inhibitors of 1,2-diacylglycerol (DG) kinase, R59022 and 1-oleyl-2-acetyl glycerol (OAG) the outward K+ current response to TRH in cells of the pituitary line GH3B6 was potentiated. This potentiation was analyzed further with the combination of microfluorimetric and electrophysiological recording techniques. Receptor-induced changes in [Ca2+]i and ion channel activation were monitored simultaneously in the same cell. It was found that R59022 and OAG altered in parallel the TRH-induced transient rise in [Ca2+]i and outward K+ current. This resulted in a significant correlation between the kinetic parameters (speed of onset, duration) of the [Ca2+]i and the K+ current responses to TRH. Intracellular application of vanadate abolished the rapid start of the TRH response presumably by its block of Ca2+ uptake into the endoplasmic reticulum, leading to depletion of a Ca2+ pool mobilizable by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The use of vanadate unmasked a slowly developing response to TRH, which was still potentiated by OAG and R59022. Together, these observations suggest that Ca2+ mobilization during the first phase of TRH action is mediated by two distinct processes, one of which is linked to receptor stimulation of DG production.

Direct evidence for control by cytosolic Ca2+ of K+ currents in pituitary cells.

Introduction Ca2+-activated K + channels are thought to play a critical role in the control of cell excitability, since they provide a link between cytosolic free calcium concentration, [Ca2+]i , and membrane potential. Here, the regulation of these channels by [Ca2+] i was studied in a pituitary cell line with the combination of two approaches: tight-seal whole-cell recording technique and microfluorimetric monitoring of [Ca2+] i with the Ca 2+ probe fura 2.

Pharmacodynamics of (DesGly10,D-Ser(t-Bu))6|[minus]|GnKH EA in 6 normal volunteers and in 2 girls with Precocious Puberty

The behaviour of [DesGly10, D-Ser(t-Bu)]6]-GnRH EA (HOE 766) has been studied in plasma and urine after intranasal (IN,200 ug) or subcutaneous (SC,10 ug/kg) administrations . A RIA for HOE 766 and its metabolites has been developed using 125I-[DesGly10, DTrp6]-GnRH EA (Gift of Dr. J. Rivier) as tracer and an antiserum to HOE 766 (gift of Dr. H. Fraser). Crossreaction with native GnRH was only 1.7 %. Sensitivity was 1 pg/tube. In 6 male adolescents, mean plasma HOE 766 concentration (±SE) was 0.46±0.08 , 0.50±0.10, 0.28±0.04, 0.24±0.04, 0.13±0.03, and 0.08 ±0.02 ng/ml, 30, 60, 90, 120 and 180 min. after IN administration. Urine excretion of HOE 766 metabolites was 9.4±2.0 ug/4h. There was a good correlation between plasma and urine levels (r=0.92). In the same 6 volunteers, plasma HOE 766 levels were 21.2±3.0, 25.9±0.8, 21.2 ±0.9, 17.1±0.7, 12.8±1.1, 8.9±0.4, and 5.9±0.8, 20, 40, 60, 120, 180 and 240 min. after the SC injection. In two girls with precocious puberty followed during 3 months with IN treatment, urine excretion of HOE 766 was in good correlation with the degree of inhibition of E2 and of LH and FSH responses to GnRH. The monitoring of HOE 766 metabolites in the urine thus appears to be helpful for the evaluation of the intranasal therapy of precocious puberty with GnRH analog.

Receptor-Stimulated Calcium Mobilization and Calcium Influx Pathways in Pituitary Cells

The importance of calcium ions (Ca”) in the control of the secretory activity of endocrine cells is well recognized. It is also known that the regulation of cytosolic free Ca2+ concentration, [Ca’+],, may involve entry of Ca’+ ions through voltageactivated Ca’+ membrane channels as well as mobilization of intracellularly sequestered Ca’+ pools. The GH3 pituitary cells, a rat clonal pituitary cell line that releases prolactin, are known to spontaneously display Ca’+ action potentials.’ We have recently demonstrated that action potentials are effective in eliciting [ &’+], transients of a magnitude sufficient to trigger [ Ca’+ ],-dependent hormone In this work, we have studied the regulation of cytosolic free Ca’+ concentration, [ Ca’+],, by the releasing factor thyrotropin-releasing hormone (TRH) at the singlecell level using tight-seal whole-cell recording techniques associated with microfluorimetric monitoring of [Ca’+],. The Ca’+ probe fura-2 was used in these experiments. GH3/B6 (GH3 subclone) cells were cultured on glass cover slides for three to five days under standard conditions. Electrophysiological recordings were obtained at room temperature using the whole-cell recording configuration of the patch-clamp technique, as described previously’; whole-cell patch pipettes (resistance 310 megohms) contained the following solution (in m M ) K+ gluconate ( l a ) , MgCl, (l), EGTA ( 1.1 ), HEPES ( S ) , pH 7.1, and fura-2 (30 pM). Buffering of [ Ca2+], obtained under these conditions is somewhat variable, such that the resting [Ca’+], values observed after equilibration of the cells with the pipette were found to vary within the range of 0.01 to 0.2 pU, Excitation light alternated rapidly (50 Hz) between ?L,

14 THE INHIBITION OF SEXUAL MATURATION BY MELATONIN IN THE MALE RAT MAY BE MEDIATED BY AMPLIFICATION OF THE OPIATERGIC NEGATIVE CONTKOL OF LH SECRETION

Our group has shown that daily administration of melatonin (MT) markedly delays sexual maturation in the male rat (Endocrinol. 112,1578,1983 and 115,2303,1984). In this study, we have evaluated the level of tonic inhibition by opiates in normal 40-day old rats, and in rats with delayed sexual development induced by daily MT (100 ug) injection between 20 and 40 days. Naloxone (NAL) s.c. injection (2.5 mg/kg) produced a significant increase of plasma LH in normal rats, not seen in MT-treated rats. Injection of morphine sulphate (MS) or of the potent Met-Enkephalin analog FK-33-824 (FK) inhibited LH secretion in control rats. In MT-treated rats, the low plasma LH levels were not affected by opiates. Pretreatment with MS, or with the FK agonist prevented the NAL-induced rise of LH in rats not treated with MT. Plasma PRL levels were decreased after NAL both in untreated- and MT-treated rats. In keeping with the observation that MT no longer inhibits sexual functions in adult rats, LH response to NAL was normal in adult rats that have been treated for 20 days with MT. These results demonstrate that MT may potentiate or mimic the tonic inhibition of LH secretion exerted by endogenous opiates during sexual development. They reinforce the concept that modulation of opiate control is important for the progress of sexual development.