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Endocrinology Vol. 141, No. 3 1017-1026
Copyright © 2000 by The Endocrine Society

ARTICLES

Differences in Gonadotropin-Releasing Hormone-Induced Calcium Signaling between Melatonin-Sensitive and Melatonin-Insensitive Neonatal Rat Gonadotrophs1

Hana Zemková and JirÍ VanecEK

Institute of Physiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic

Address all correspondence and requests for reprints to: Hana Zemková, Institute of Physiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic. E-mail: zemkova{at}biomed.cas.cz


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The sensitivity of GnRH-stimulated calcium signaling to melatonin, in a subpopulation of neonatal gonadotrophs, is supposed to be attributable to melatonin receptors. However, it is not yet known whether the intracellular pathway for GnRH action in melatonin-sensitive cells is the same as in melatonin-insensitive cells. By monitoring intracellular Ca2+ changes as an outward current carried through apamin-sensitive Ca2+-activated K+ channels, we compared GnRH-induced calcium responses in these two subpopulations of neonatal gonadotrophs. GnRH induced various oscillatory, as well as nonoscillatory, responses in both cell types that was not related to melatonin sensitivity. Melatonin-sensitive GnRH-induced responses could be clearly distinguished according to the pharmacological properties of their latency. The latency increased in zero extracellular Ca2+ or with the addition of nifedipine, staurosporine, and ryanodine. This effect was only rarely observed in melatonin-insensitive cells. This indicates that there are two pathways for initiation of GnRH-induced calcium signaling in neonatal gonadotrophs. The first pathway is mediated by inositol 1,4,5,-trisphosphate production, whereas the second involves extracellular calcium entry through voltage-dependent L-type Ca2+ channels, protein kinase C activation, and Ca2+ release from a ryanodine-sensitive store, which may coactivate Ca2+ release from an inositol 1,4,5,-trisphosphate-sensitive store. Only the second mechanism is accessible to inhibition by melatonin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RELEASE OF HORMONES LH and FSH from pituitary gonadotrophs are controlled by GnRH, which induces an increase in intracellular Ca2+ concentration ([Ca2+]i). In adult gonadotrophs, activation of GnRH receptor is coupled to a pertussis toxin-insensitive G protein that stimulates phospholipase C. This leads to hydrolysis of membrane phosphoinositides into inositol 1,4,5,-trisphosphate (InsP3) and diacylglycerol (for review, see Refs. 1, 2, 3, 4). The InsP3 initiates oscillatory release of Ca2+ from intracellular stores by binding to its specific receptor in the endoplasmic reticulum (5). Diacylglycerol activates Ca2+-dependent protein kinase C (6) which apparently modulates extracellular Ca2+ entry via voltage-dependent calcium channels. The GnRH-stimulated intracellular Ca2+ signaling pathway in neonatal gonadotrophs is supposed to be similar to that in adults (7), but it has not yet been studied in detail. Immature gonadotrophs exhibit some specific properties: they are larger and morphologically more heterogeneous, their storage granules contain more often only LH or FSH, whereas that of adult gonadotrophs usually contain both (8). Moreover, the GnRH-stimulated LH release, as well as [Ca2+]i increase, in a subpopulation of neonatal gonadotrophs is inhibited by the pineal hormone melatonin (9). Melatonin is supposed to reduce the GnRH-induced [Ca2+]i increase by inhibiting both extracellular Ca2+ entry (10) and Ca2+ release from intracellular stores (11). The intracellular mechanism of its action is not yet fully understood (12). Melatonin receptor, coupled to pertussis toxin-sensitive G protein (9, 13), gradually disappears from the pituitary in the course of postnatal development. Nevertheless, it persists even in the adult pituitary, although its concentration is only about one tenth of the neonatal value (14). This raises the question of whether the presence of melatonin receptor is the only factor responsible for a specific melatonin sensitivity of neonatal gonadotrophs. The aim of the present study was to compare GnRH-induced intracellular calcium oscillations in melatonin-sensitive and melatonin-insensitive neonatal gonadotrophs. This could help to understand the developmental changes in intracellular GnRH-induced calcium signaling, as well as the mechanism of action of melatonin. By monitoring [Ca2+]i transients as oscillatory outward apamin-sensitive Ca2+-activated K+ currents (11, 15, 16), we found that there are two different pathways for GnRH-stimulated intracellular Ca2+ signaling in neonatal gonadotrophs and that only one of these pathways is sensitive to melatonin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell preparation and culture
Primary dissociated pituitary cultures were prepared from female rat pups kept with their mothers under an automatically regulated light-dark cycle (12-h light, 12-h dark; lights on at 0600 h) from the age of 2 days. Six- to 10-day-old pups were killed by decapitation between 0830 and 1000 h. The anterior pituitary glands were rapidly removed under sterile conditions, gently disrupted, and dissociated by papain (17). The dispersed cells were separated from the cell debris and cell clusters using a discontinuous albumin gradient and then resuspended in Eagle’s MEM (10). The dissociated cells were counted, diluted to 0.6 x 106 cells/ml MEM, and seeded onto the bottom of 35-mm culture dishes covered with poly-L-lysine. The pituitary cells were cultured in MEM, supplemented with 5% neonatal rat serum from the pituitary donors + 5% FCS in air/CO2 at 37 C. The cultures were used for experiments within 1–2 days after dissection.

Patch-clamp recordings
Changes in [Ca2+]i were monitored in voltage-clamped cells by recording currents through apamin-sensitive Ca2+-activated K+ channels (11, 15, 16). Perforated patch-clamp (18) and standard whole-cell technique (19) were used. Patch electrodes were pulled from glass tubes 1.65 mm in od. The tip of the pipette had an od of about 2 µm, and the pipette resistance was 4–10 M{Omega}. When nystatin was present in the pipette (see Solutions), 10 min after seal formation the perforated seal resistance was 28.1 ± 7.2 M{Omega} (n = 15), and the mean capacitance of the cells was 4.1 ±1.5 pF (n = 16). No series resistance compensation was used. The plasma membrane potential was held at -40 mV. The correction for the Donnan potential, because of the absence of impermeant anions in the pipette solution (see Solutions) and liquid junction potential, were ignored. Membrane currents were recorded with an Axopatch-1D amplifier (Axon Instruments, Foster City, CA).

Solutions
During the experiments, dishes with cell cultures were continuously superfused with an extracellular solution of the following composition (in mM): 160 NaCl, 2.5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES; pH adjusted with 1 M NaOH to 7.3. In a Ca2+-deficient medium, CaCl2 was omitted and, in some experiments, 5 mM EGTA added. Patch electrodes used for perforated patch-clamp recordings were filled with an intracellular solution containing (in mM): 140 KCl, 1 MgCl2, and 10 HEPES; pH adjusted with 1 M KOH to 7.2. Nystatin and dispersing agent Pluronic F-127 were added to the intracellular solution from stock solutions in dimethylsulfoxide to obtain a final concentration of 250 µg/ml nystatin and 500 µg/ml Pluronic. To evoke InsP3-induced oscillations, 2 mM ATP, 0.3 mM GTP, and 10 µM InsP3 were added to the intracellular solution without nystatin.

Drug application
Control and drug-containing solutions were applied with a fast gravity driven perfusion system consisting of an array of 10 glass tubes, each approximately 400 µm in diameter. Movement of the glass tube array and solution application were controlled by a step motor and miniature Teflon solenoid valves governed by microcomputer. A complete change of the solution around a cell took less than 100 msec. During the recording from a single cell, GnRH was repeatedly applied at 60- to 120-sec intervals. Our results are based on data from cells that exhibited recovery of GnRH-induced responses after inhibition or modulation.

Data storage and analysis
Data were stored in digital form using a modified digital-audio processor (Sony PCM-501ES, frequency 20 kHz) and a video tape recorder. The currents were filtered at 50 Hz and sampled at 100 Hz. The amplitude measured at the maximum peak current, the time to onset, and frequency of oscillatory currents induced by GnRH were analyzed using pClamp 6.0.2. software (Axon Instruments). Data are expressed as the mean ± SEM; n represents the number of cells; P < 0.01 was considered significant.

Chemicals
Caffeine, EGTA, GnRH, melatonin, nifedipine, nystatin, ryanodine, staurosporine, and D-myo-inositol 1,4,5,-Tris-phosphate potassium salt were obtained from Sigma (St. Louis, MO); U-73122 and U-73343 from Calbiochem (La Jolla, CA); and Pluronic F-127 from Molecular Probes, Inc. (Eugene, OR). The experiments were done at room temperature (20–25 C).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Different oscillatory patterns of GnRH-induced Ca2+-dependent K+ currents (IK(Ca)) in neonatal gonadotrophs and their sensitivity to melatonin
In neonatal gonadotrophs voltage-clamped at -40 mV, GnRH induced various types of outward IK(Ca). The oscillatory pattern of these responses differed considerably from cell to cell but was very similar when the cell was stimulated repeatedly with the same dose of GnRH. This phenomenon, called Ca2+ fingerprint (20), has been also found in other cell types and is supposed to reflect the specific intrinsic properties of each cell (21). In spite of this cell-specific variability, several types of GnRH-induced IK(Ca) responses can be distinguished in neonatal gonadotrophs according to both the presence or absence of baseline oscillations and to the concentration dependence of the amplitude or frequency of oscillations.

In 15 of 18 neonatal gonadotrophs exhibiting GnRH-induced IK(Ca) baseline oscillations, i.e. oscillations in which the current reached the baseline between individual spikes, the frequency of oscillations increased with GnRH (Fig. 1Go, A and B). In 3 remaining cells, the frequency of baseline oscillations was constant and independent of GnRH concentration (Fig. 1CGo). In melatonin-sensitive cells with frequency-modulated oscillations (n = 6), an increase in GnRH from 1 to 30 nM significantly increased the frequency of baseline oscillations from 13.8 ± 1.0 min-1 to 29.4 ± 6.1 min-1 (P < 0.01; Fig. 1AGo) but had little effect on the maximum peak current amplitude. This increased from 66 ± 11 pA to 83 ± 17 pA (P > 0.01). The latency preceding the GnRH-induced response, measured from the beginning of GnRH application to the first sign of the response, decreased by 26 sec (from 33.5 ± 4.7 sec to 7.4 ± 0.6 sec; P < 0.01). An increase in GnRH concentration above 10 nM produced a transition in the response from baseline oscillations to a biphasic state consisting of an initial nonoscillatory spike, with or without superimposed oscillations, followed by baseline oscillations (7, 22, 23). The frequency of superimposed oscillations was lower than that of baseline oscillations, but it did not change with increasing GnRH; its average value was 12.5 ± 2.7 min-1 (Fig. 1AGo, triangles). Melatonin (1 nM), applied together with GnRH, significantly (P < 0.01) prolonged the latency period by 7–20 sec at all GnRH concentrations tested but had little or no effect on the response amplitude or frequency of baseline oscillations (Fig. 1AGo). If applied during GnRH stimulation, melatonin attenuated the amplitude and eventually stopped ongoing oscillations (see Figs. 4AGo and 5BGo). Nevertheless, this inhibition was only transient; in the presence of melatonin the oscillations spontaneously reappeared after 10–40 sec of inhibition (see Fig. 6BGo). In melatonin-insensitive cells with frequency-modulated baseline oscillations (n = 9; Fig. 1BGo), an increase in GnRH from 1 to 30 nM increased the frequency of baseline oscillations from 19.3 ± 1.9 min-1 to 28.0 ± 2.8 min-1 (P < 0.01). This was similar to results in melatonin-sensitive cells (Fig. 1AGo). Increasing GnRH had no effect on the maximum peak current amplitude (58 ± 16 pA at 1 nM GnRH and 65 ± 7 pA at 30 nM GnRH, P > 0.01). The concentration dependence of the latency was much lower in melatonin-insensitive cells than in melatonin-sensitive cells; an increase in GnRH concentration from 1 to 30 nM reduced the latency by only 10 sec (from 16.2 ± 6.2 sec to 6.1 ± 0.8 sec). Melatonin-insensitive cells also exhibited biphasic responses at higher GnRH concentrations. The frequency of oscillations superimposed on an initial spike was constant (15.2 ± 1.2 min-1; Fig. 1BGo, triangles).



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  		Figure 1. GnRH-induced IK(Ca) responses with baseline oscillations in melatonin-sensitive and melatonin-insensitive neonatal gonadotrophs. A, Responses to 1, 10, and 30 nM GnRH recorded from melatonin-sensitive cell in the presence or absence of 1 nM melatonin. The time of GnRH and melatonin application is indicated by horizontal bars. GnRH dose-response graphs show the current amplitude (in percent of maximum current induced by high GnRH concentrations),the frequency of oscillations (number of spikes per 1 min), and latency (the time from the beginning of GnRH application to the first spike) before (closed symbols) and after (open symbols) melatonin application. Triangles in the frequency graphs refer to the initial superimposed oscillations in biphasic responses. Each point represents the mean ± SEM from six cells. B, GnRH-induced responses recorded in the presence or absence of melatonin from melatonin-insensitive cell. The graphs show the current amplitude, frequency of oscillations, and latency of GnRH-induced responses obtained from nine cells. C, An example of melatonin-sensitive baseline oscillation, the frequency of which was independent of GnRH concentration. All cells were voltage-clamped to -40 mV.

 


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  		Figure 4. The effect of Ca2+-deficient solution on GnRH-induced responses in melatonin-sensitive and melatonin-insensitive gonadotrophs. A, Latency prolongation by melatonin (1 nM) and by extracellular Ca2+ omission (0 Ca2+) in a melatonin-sensitive response with baseline oscillations induced by 10 nM GnRH. Melatonin inhibited ongoing oscillations in the presence and in the absence of extracellular Ca2+. Contrary to melatonin, the Ca2+-deficient solution applied during GnRH stimulation did not inhibit ongoing oscillations. B, Latency prolongation by the Ca2+-deficient solution in melatonin-sensitive GnRH-induced response without baseline oscillations. Melatonin entirely inhibited the response if applied together with GnRH and during GnRH stimulation. In Ca2+-deficient solution, melatonin also inhibited the response during GnRH stimulation, but the GnRH-induced response did not recover after melatonin removal. C, The lack of any effect of melatonin and Ca2+-free solution (in the presence of 5 mM EGTA) in a melatonin-insensitive cell with baseline oscillations.

 


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  		Figure 5. The effect of the holding potential (HP) on the amplitude and latency of GnRH-induced IK(Ca) responses in melatonin-insensitive (A) and melatonin-sensitive (B) cells. The maximum amplitude of GnRH-induced IK(Ca) decreases at negative holding potentials. Its reversal potential (Erev= -91 ± 6 mV, n = 6) was near to the calculated reversal potential for K+ ions (-96 mV) both in melatonin-sensitive (open symbols) and melatonin-insensitive (closed symbols) cells (C). On the other hand, voltage dependence of the latency was observed only in melatonin-sensitive cells (D).

 


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  		Figure 6. The effect of ryanodine on GnRH-induced responses. A, An example of nonoscillatory GnRH-induced response in melatonin-sensitive cell. This response was entirely inhibited by melatonin (1 nM), and its latency was prolonged by ryanodine (2 µM). B, Transient inhibition of ongoing GnRH-induced baseline oscillations by melatonin and ryanodine. C, Lack of any effect of ryanodine in melatonin-insensitive cell exhibiting nonoscillatory GnRH-induced response.

 
In 3 of 18 tested neonatal gonadotrophs, GnRH induced unusually large baseline oscillations, the frequency of which did not increase with GnRH concentration (Fig. 1CGo). The threshold concentration was always above 3 nM. An increase in GnRH from 10 nM to 100 nM increased the amplitude from 140 ± 31 pA to 176 ± 35 pA (P > 0.01) and shortened the latency by 9.9 sec (from 19.5 ± 8.9 sec to 9.6 ± 2.4 sec) (P > 0.01). The frequency of oscillations was 11.2 ± 2.4 min-1 at 10 nM GnRH and 12.7 ± 3.0 min-1 at 100 nM GnRH. No biphasic responses were observed in these cells, even at very high GnRH doses. Melatonin, if applied together with GnRH, prolonged the latency without any effect on the frequency or amplitude of oscillations in 2 of these cells (not shown). If applied during ongoing oscillations, melatonin decreased the amplitude and eventually stopped oscillations (Fig. 1CGo).

GnRH-induced IK(Ca) responses without baseline oscillations were either nonoscillatory or with superimposed oscillations of low frequency that varied from 2 to 11 min-1 (Fig. 2Go). In melatonin-sensitive cells (n = 5; Fig. 2AGo), an increase in GnRH from 3 to 30 nM significantly increased the maximum peak current amplitude from 34 ± 9 pA to 63 ± 22 pA (P < 0.01) and shortened the latency by 21.5 sec (from 35.2 ± 12.9 sec to 13.7 ± 3.7 sec; P < 0.01). The superimposed oscillations were usually present only at lower GnRH doses, and their frequency (7.5 ± 1.9 min-1) was independent of the agonist concentration. Melatonin (1 nM) inhibited responses induced by 3 nM GnRH in 3 of 5 cells and reduced the amplitude in the remaining 2 cells. Melatonin only prolonged the latency of responses induced by higher GnRH concentration. If applied during GnRH stimulation, melatonin reduced the amplitude or inhibited the response (see Fig. 4BGo), but no spontaneous recovery of the current was observed in the continuous presence of melatonin (not shown). In melatonin-insensitive cells (n = 3; Fig. 2BGo), an increase in GnRH concentration from 3 to 30 nM also increased the maximum current amplitude from 59 ± 12 to 121 ± 20 pA (P < 0.01); the latency was shortened only by 1.7 sec (from 6.2 ± 1.0 sec to 4.5 ± 0.6 sec). The mean frequency of superimposed oscillations, which were rare and observed only at lower GnRH, was 6.5 ± 2.1 min-1. These results provide evidence that neonatal gonadotrophs exhibit separate amplitude-modulated nonoscillatory responses, with or without superimposed low-frequency oscillations; these are not attributable to summation of high-frequency baseline oscillations.



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  		Figure 2. GnRH-induced IK(Ca) responses without baseline oscillations in melatonin-sensitive (A) and melatonin-insensitive (B) cells. The time of GnRH and melatonin (1 nM) applications is indicated by horizontal bars. GnRH dose-response graphs show the current amplitude and latency before (closed symbols) and after (open symbols) melatonin application (for details see legend to Fig. 2Go). Each point represents the mean ± SEM from three to six cells.

 
The fraction of responses with and without baseline oscillations in melatonin-sensitive and melatonin-insensitive cells was determined in a large population of neonatal gonadotrophs (n = 268) stimulated with 10 nM GnRH (Fig. 3Go). In 70% of these cells, GnRH induced IK(Ca) responses with baseline oscillations; in the remaining 30% it induced responses without baseline oscillations. Melatonin (1 nM), applied together with GnRH, prolonged the latency in 52% of cells with baseline oscillations and entirely inhibited 7% of these cells (Fig. 3AGo). Frequencies of baseline oscillations induced by 10 nM GnRH varied from 6 to 38 min-1, both in melatonin-sensitive and melatonin-insensitive cells (Fig. 3BGo). However, melatonin-sensitive cells exhibited baseline oscillations of lower frequencies (from 6 to about 16 min-1) more frequently than melatonin-insensitive cells. In gonadotrophs exhibiting GnRH-induced responses without baseline oscillations, melatonin prolonged the latency in 31% of cells and entirely inhibited responses in another 38% (Fig. 1AGo). This indicates that GnRH-induced responses without baseline oscillations are more accessible to complete inhibition by melatonin. Nevertheless, melatonin sensitivity of both response types is very similar; 59% of GnRH responses with baseline oscillations and 69% of responses without baseline oscillations were sensitive to melatonin.



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  		Figure 3. A, Comparison of melatonin sensitivity between GnRH-induced responses with and without baseline oscillations. The columns refer to the fractions of melatonin-insensitive cells (Mel-) and melatonin-sensitive cells (Mel+) in which melatonin (1 nM) applied together with GnRH (10 nM) prolonged the latency or entirely inhibited the GnRH-induced responses. B, Distribution histograms of baseline oscillation frequencies induced by 10 nM GnRH in melatonin-insensitive (open columns; n = 77) and melatonin-sensitive (closed columns; n = 107) cells.

 
Extracellular Ca2+ dependence of latency, the effect of nifedipine and membrane potential
Dose-response experiments described above showed that it is not the oscillatory pattern of GnRH-induced responses but rather the duration of the latency that is different in melatonin-sensitive and melatonin-insensitive neonatal gonadotrophs. The latency for neonatal GnRH-induced calcium responses is prolonged in the absence of extracellular Ca2+ (11), but it has not been found in adult gonadotrophs (1, 16, 22, 24, 25, 26). We noticed that the latency of neonatal GnRH-induced responses is not always prolonged in Ca2+-deficient medium and that this phenomenon is related to melatonin sensitivity. In all melatonin-sensitive cells tested (n = 13), short-lasting external Ca2+ omission for 10–60 sec before and during GnRH application prolonged the latency as did melatonin (by 19–21 sec), but it had no effect on the response amplitude or frequency of oscillations (Table 1Go; Fig. 4Go, A and B). Melatonin applied during GnRH stimulation inhibited ongoing responses in the presence, as well as in the absence, of extracellular Ca2+ but the external Ca2+ removal had no effect on ongoing oscillations (Fig. 4AGo). External Ca2+ removal prolonged the latency by 13.9 sec (from 7.5 ± 1.1 sec to 21.4 ± 6.5 sec) in 5 of 15 melatonin-insensitive cells tested but in the remaining 10 melatonin-insensitive cells, it had no effect (Fig. 4CGo).


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  		Table 1. Different pharmacology of two basic types of GnRH-induced IK(Ca) responses in melatonin-sensitive neonatal gonadotrophs

 
Latency prolongation was also observed in the presence of the dihydropyridine calcium channel antagonist, nifedipine. If applied together with GnRH (10 µM), nifedipine (2 µM) prolonged the latency by 26–31 sec without any effect on the amplitude or frequency of baseline oscillations in six of eight melatonin-sensitive cells (Table 1Go). If applied during GnRH stimulation, nifedipine inhibited ongoing oscillations or nonoscillatory response amplitude as did melatonin (not shown). No effect of nifedipine was observed in six melatonin-insensitive cells.

This indicates that GnRH stimulates external Ca2+ entry during the latency period in melatonin-sensitive neonatal gonadotrophs and that this Ca2+ entry is mediated most probably by L-type voltage-dependent Ca2+ channels. If so, the latency in melatonin-sensitive cells should depend on membrane potential. Fig. 5Go shows that the amplitude of GnRH-induced outward current increased at holding potentials ranging from positive to -40 mV, but it decreased at holding potentials negative to -40 mV. Its reversal potential was near to the calculated reversal potential for K+ (Fig. 5CGo). The voltage-dependence of the IK(Ca) amplitude was identical in melatonin-sensitive and in melatonin-insensitive cells. On the other hand, the latency that only slightly increased in melatonin-insensitive cells (from 3.7 ± 1.0 sec at -30 mV to 6.7 ± 2.5 sec at -80 mV; P > 0.01) increased significantly (from 6.0 ± 2.5 sec at -30 mV to 19.5 ± 5.7 sec at -80 mV; P < 0.01) in melatonin-sensitive cells (Fig. 5DGo). Depolarization itself, in the absence of GnRH, evoked no response in both melatonin-sensitive and -insensitive cells.

The effect of ryanodine, caffeine, and staurosporine
The extracellular Ca2+ sensitivity of the latency in melatonin-sensitive cells indicated that Ca2+-induced calcium release from internal stores controlled by the ryanodine receptor could be involved in the response to GnRH. Ryanodine (2 µM), applied together with GnRH (10 nM), prevented the response in 3 of 17 melatonin-sensitive cells and prolonged the latency by 25–31 sec in the remaining 14 cells (Table 1Go, Fig. 6AGo). Ryanodine was without any effect on the amplitude or frequency of oscillations. When applied during GnRH stimulation, ryanodine transiently inhibited ongoing baseline oscillations similar to melatonin (Fig. 6BGo) or lowered the amplitude of nonoscillatory responses (not shown). No effect of ryanodine was observed in 3 of 5 melatonin-insensitive cells (Fig. 6CGo). Caffeine (10 mM), another modulator of the ryanodine receptor, applied alone did not induce any response in neonatal gonadotrophs (n = 10). If it was applied with GnRH, it shortened the latency in melatonin-sensitive cells (n = 6) without affecting the response amplitude or frequency of baseline oscillations (Tale 1). No effect of caffeine was found in 3 melatonin-insensitive cells.

Secondary to GnRH-induced stimulation of phospholipase C is activation of protein kinase C. Its role in intracellular calcium signaling in adult gonadotrophs is not yet fully established, but it is supposed to modulate depolarization-stimulated extracellular Ca2+ entry responsible for refilling of InsP3-sensitive Ca2+ stores (27, 28). We found that the protein kinase C inhibitor staurosporine (1 nM) (29) prolonged the latency of GnRH-induced responses by 26–28 sec in all melatonin-sensitive cells (n = 10) without significant effect on the response amplitude or frequency of oscillations (Fig. 7Go, A and B; and Table 1Go). Staurosporine, applied during GnRH stimulation, transiently inhibited ongoing oscillations (not shown) in a manner similar to that of melatonin, nifedipine or ryanodine and it reduced the amplitude of responses without baseline oscillations. No effect of staurosporine was observed in 12 of 13 melatonin-insensitive cells (Fig. 7CGo).



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  		Figure 7. The effect of protein kinase C inhibitor, staurosporine, on GnRH-induced responses. In melatonin-sensitive cells, the latency of both baseline oscillations (A) and nonoscillatory responses (B) induced by GnRH (10 nM) was prolonged by melatonin (1 nM) and by staurosporine (10 nM). No effect of staurosporine was observed in melatonin-insensitive cell (C).

 
Specific pharmacological sensitivity of GnRH-induced responses in melatonin-sensitive neonatal gonadotrophs was not a constant property of these cells. In melatonin- and drug-insensitive cells recorded from for a longer time, GnRH-induced responses became spontaneously melatonin- and drug-sensitive after 15–30 min (Fig. 8Go). This time-dependent change was found in 9 of 27 melatonin-insensitive cells recorded for at least 35 min. The opposite effect, e.g. transformation from melatonin-sensitive to melatonin-insensitive state, was never observed.



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  		Figure 8. Time-dependent changes in pharmacology of GnRH-induced responses. GnRH-induced responses were insensitive to melatonin (1 nM), ryanodine (2 µM), and nifedipine (2 µM) at the beginning of patch clamp recording (Control) and they spontaneously changed into melatonin- and drug-sensitive state after about 15 min.

 
InsP3-induced oscillations in neonatal gonadotrophs
To determine whether all types of GnRH-induced calcium responses in neonatal gonadotrophs are attributable to Ca2+ release from InsP3-sensitive stores and whether the responses induced directly by InsP3 are sensitive to melatonin, we induced IK(Ca) responses by intracellular application of InsP3. With a saturating concentration of 10 µM InsP3 in the pipette, the InsP3-stimulated responses were observed immediately after whole-cell formation in 35 of 68 pituitary cells (Fig. 9Go). Theoretically, only about 20% of pituitary cells (i.e. 14 of 68 cells) could be gonadotrophs, which indicates that some other cell types have been also tested. Most frequently, InsP3-induced responses were nonoscillatory currents lasting for 30–60 sec. These were observed in 22 of InsP3-sensitive cells (Fig. 9AGo). The maximum amplitude of these responses was 83 ± 15 pA; they did not have superimposed oscillations. The remaining 13 InsP3-sensitive cells exhibited biphasic responses or baseline oscillations lasting for up to 5 min (Fig. 9Go, B and C). The maximum peak current amplitude of these responses was 65 ± 17 pA, and the frequency of oscillations ranged from 11–29 min-1 (mean = 16.3 ± 1.1 min-1). Addition of melatonin, ryanodine (Fig. 9Go, B and C), staurosporine, nifedipine, or extracellular Ca2+ omission (not shown) was without any effect. Extracellular application of GnRH had no or little effect on the amplitude or frequency of ongoing InsP3-induced oscillations (Fig. 9CGo). In 6 of 33 cells in which intracellular InsP3 did not induce any response, extracellular GnRH evoked large low-frequency oscillations (Fig. 9DGo) that were inhibited by melatonin (Fig. 9EGo). This indicates that InsP3-induced oscillations are insensitive to melatonin and that not all neonatal GnRH-induced responses are mediated by InsP3. Moreover, these experiments provided evidence that the composition of intracellular solution in the pipette was sufficient to permit melatonin inhibition.



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  		Figure 9. InsP3-induced IK(Ca) responses in neonatal pituitary cells and their insensitivity to melatonin. Intracellular solution containing 10 µM InsP3 was applied into the cell during breaking through to the whole-cell from the cell-attached mode (indicated by arrow). Nonoscillatory responses (A) and biphasic responses or baseline oscillations (B and C) were induced. InsP3-induced responses were insensitive to melatonin (1 nM) or ryanodine (2 µM). In InsP3-insensitive pituitary cells, extracellular GnRH application induced low-frequency baseline oscillations (D and E), which could be inhibited by melatonin.

 
Ineffectiveness of U-73122 as a specific phospholipase C blocker in neonatal gonadotroph.
The importance of InsP3 in generation of GnRH-induced [Ca2+]i responses was also examined by testing the effect of aminosteroid U-73122, a specific phospholipase C inhibitor (30). We found that U-73122 (2–10 µM) applied for 60–20 sec before and during GnRH stimulation had no effect on the amplitude and frequency of GnRH-induced responses, but it prolonged the latency in all melatonin-sensitive cells (Table 1Go). Nevertheless, its structural inactive analog, U-73343, had an effect similar to that of U-73122 (Table 1Go). Both aminosteroids were ineffective in melatonin-insensitive cells (n = 7). This means that U-73122 cannot be considered as a specific inhibitor of phospholipase C in neonatal gonadotrophs and/or that phospholipase C is not involved in neonatal GnRH-induced calcium signaling.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The mechanism of GnRH-induced [Ca2+]i oscillations is relatively well established in gonadotrophs from adult rats (for review, see Refs. 1, 2, 3, 4), but little is known about GnRH-induced intracellular Ca2+ signaling in immature gonadotrophs. Using an electrophysiological approach, we found that gonadotrophs from 6- to 10-day-old rats exhibit three basic types of GnRH-induced calcium responses that can be distinguished by presence or absence of baseline oscillations and by the concentration dependence of the frequency of oscillations. These responses are: 1) baseline oscillations with frequency dependent on agonist concentration; 2) low-frequency oscillations, either baseline or superimposed on a nonoscillatory response with the frequency independent of agonist concentration; and 3) amplitude-modulated nonoscillatory responses. The frequency-modulated baseline oscillations (response 1), observed in the majority of neonatal gonadotrophs, are also the main type of GnRH-induced [Ca2+]i responses in adult gonadotrophs (7, 22, 24, 25, 26, 31). These responses could be simulated by intracellular application of InsP3 (16, 32), indicating that they are caused by Ca2+ release from InsP3 receptor-controlled intracellular Ca2+ stores. The GnRH-induced constant-frequency oscillations (response 2), baseline or superimposed on nonoscillatory current, were not induced by intracellular InsP3 application. They are similar to nonreceptor oscillations that can be stimulated in adult gonadotrophs only by thapsigargin (33). Thapsigargin-induced oscillations are supposed to be mediated by capacitative Ca2+ entry (34) and maintained by its slow feedback inhibition (35, 36). The mechanism by which GnRH stimulates these oscillations in neonatal gonadotrophs is not clear. The superimposed oscillations also resemble sinusoidal oscillations observed in many cell types. Sinusoidal oscillations are supposed to be caused by fluctuating levels of InsP3 caused by a negative-feedback effect of protein kinase C on phospholipase C activity (37). In this case, the superimposed oscillations should be eliminated by inhibition of protein kinase C, but this has not been observed in neonatal gonadotrophs. Nonoscillatory amplitude-modulated GnRH-induced responses without baseline oscillations (response 3) have not yet been described in adult gonadotrophs. Nevertheless, it is possible that they are identical with initial large nonoscillatory spikes regularly present at the beginning of biphasic responses induced by higher GnRH concentrations in adult, as well as in neonatal, gonadotrophs. The amplitude of this initial spike of biphasic responses is also dependent on GnRH concentration (23), and both nonoscillatory and biphasic responses could be induced by intracellular InsP3 application.

Melatonin inhibited or prolonged the latency in about 60–70% of all types of neonatal responses. It indicates that melatonin sensitivity of neonatal gonadotrophs is independent of the oscillatory pattern of their GnRH-induced responses.

Melatonin-sensitive GnRH responses could be clearly distinguished according to the pharmacological properties of their latency. In melatonin-sensitive neonatal gonadotrophs, the latency was prolonged in Ca2+-deficient medium or in the presence of nifedipine, ryanodine, and staurosporine. However, this effect was rare or lacking in melatonin-insensitive neonatal gonadotrophs. No effect of ryanodine, external Ca2+ removal, and staurosporine on the nitiation phase of GnRH-induced [Ca2+]i responses has been also observed in melatonin-insensitive adult gonadotrophs (1, 16, 22, 24, 25, 28, 32, 33). This indicates that extracellular Ca2+ entry through dihydropyridine-sensitive Ca2+ channels, protein kinase C activation, and calcium release from ryanodine-sensitive store are involved in initiation of GnRH-induced responses in a melatonin-sensitive subpopulation of neonatal gonadotrophs. For agonist-stimulated InsP3-mediated [Ca2+]i responses, the latency to the first [Ca2+]i spike reflects the time necessary for InsP3 generation (21). The latency of GnRH-induced response was inversely related to the GnRH concentration in all neonatal gonadotrophs, but this dependence was much more evident in melatonin-sensitive cells. This observation also indicates that the mechanisms of GnRH-induced [Ca2+]i response initiation might be different in melatonin-sensitive and melatonin-insensitive cells.

Dihydropyridine-sensitive L-type Ca2+ channels are known to be expressed in adult gonadotrophs (1, 16, 22, 24, 25, 26, 32, 38, 39). These voltage-dependent Ca2+ channels could be active in our experiments in which the cells were voltage-clamped to -40 mV. Furthermore, the latency of GnRH-induced responses in melatonin-sensitive cells was prolonged at more negative membrane potentials. GnRH could activate these channels by modulation of channel protein by activation of protein kinase C, reported in gonadotrope lineage {alpha}T3–1 cells (40). Another possibility is direct activation of L-type Ca2+ channels by ß{gamma} subunit released from activated G protein, found in many other cell types (41).

The ryanodine receptor has not yet been described in gonadotrophs, but it has been found in anterior pituitary GH3 cells (42). Both InsP3 and ryanodine receptors coexist and interact in the same cell of many mammalian organs (43, 44, 45, 46). In these systems, calcium released from the ryanodine receptor-controlled stores always contributed to an alteration of cytoplasmic calcium levels. However, this has not been found in neonatal gonadotrophs. According to our finding, ryanodine only prolonged the latency but did not induce any significant changes in the amplitude of GnRH-induced responses if applied together with GnRH. It only transiently inhibited ongoing oscillations if applied during GnRH stimulation. Also caffeine, if applied alone, did not evoke any response but shortened the latency if applied with GnRH. It is possible that Ca2+-induced calcium release from ryano-dine-sensitive stores in neonatal gonadotrophs is very small but is sufficient to coactivate an InsP3 receptor-controlled Ca2+ channel (43). We thus assume that GnRH-induced Ca2+ entry during the latency period may stimulate Ca2+ release from ryanodine-sensitive stores. This would sensitize the InsP3 receptor in melatonin-sensitive cells. The sensitization of calcium release from InsP3-sensitive stores might be necessary under conditions when neonatal GnRH-induced production of InsP3 is low. In melatonin-insensitive cells, the initiation step in the GnRH-induced calcium signaling pathway involves only InsP3 production that is sufficient to initiate calcium release from InsP3-sensitive stores.

It is generally accepted that Ca2+ release from ryanodine-sensitive stores in nonmuscle cells is activated not only by Ca2+ influx but also by some second messenger (47). It implies that GnRH could stimulate parallel production of two second messengers to activate both the InsP3 and ryanodine receptors in melatonin-sensitive neonatal gonadotrophs. Changes in melatonin and drug sensitivity observed in some neonatal gonadotrophs indicate that two second messengers might be produced both in melatonin-sensitive and melatonin-insensitive cells. The reason for the time-dependent change in melatonin sensitivity might be a translocation of internal Ca2+ stores within the cell (46) during prolonged patch clamp recording. This could change protein-protein interaction between plasma membrane Ca2+ channels and internal Ca2+ stores (48, 49).

Melatonin had no effect on the InsP3-induced responses, and its effect on GnRH-induced responses was similar to that of external Ca2+ removal, ryanodine, staurosporine and nifedipine. This indicates that the potential target for melatonin, in the cascade of GnRH receptor-mediated signaling events, is localized upstream from the InsP3 receptor activation. It could include activated G protein, phospholipase C, protein kinase C, Ca2+ channel and ryanodine receptor. Inhibition of extracellular Ca2+ entry as the primary step of melatonin action seems to be unlikely because melatonin inhibits GnRH-induced calcium responses also in the absence of extracellular calcium (11). Melatonin has been shown to decrease GnRH-induced formation of diacylglycerol (50), indicating that it could act through inhibition of phospholipase C. Nevertheless, melatonin substantially prolonged the latency without significant effect on the amplitude or frequency of GnRH-induced oscillations, indicating that the InsP3 production also might not be affected. Moreover, our experiments did no confirm the involvement of phospholipase C in neonatal GnRH-induced [Ca2+]i signaling. Melatonin thus could act at the level of G protein, protein kinase C, or ryanodine receptor but at present, we cannot precisely localize the site of its action.

In conclusion, we found that there are two pharmacologically distinguished intracellular mechanisms for GnRH-induced calcium signaling in neonatal gonadotrophs. They differ in the initial steps leading to stimulation of Ca2+ release from InsP3-sensitive stores. The first pathway involves GnRH-induced InsP3 production, whereas the second pathway involves protein kinase C activation, external Ca2+ influx through voltage-sensitive L-type Ca2+ channel, and Ca2+ release from a ryanodine-sensitive store that apparently sensitizes the InsP3 receptor. The first mechanism is insensitive to melatonin, but the second mechanism is accessible to inhibition by melatonin.


   Acknowledgments
 
We would like to thank Pavel Hnik of the Institute of Physiology Academy of Sciences of the Czech Republic and Richard Orkand of the University of Puerto Rico for reading the manuscript.


   Footnotes
 
1 This research was supported by the Grant Agency of the Czech Republic, Grants No. 309/97/0513 and 309/99/0215, by Internal Grant Agency of Academy of Sciences, Grant No. A 50 11 705, and by the Ministery of Education, Youth and Sport of the Czech Republic, Grant No. 97 099. Back

Received June 7, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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