J. Steroid Biochem. Molec. Biol. Vol. 41, No. 3-8, pp. 453-467, 1992 Printed in Great Britain. All fights reserved
CALCIUM
SIGNALING
AGONIST-STIMULATED
AND
0960-0760/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press plc
SECRETORY
PITUITARY
RESPONSES
IN
GONADOTROPHS
STANKO S. STOJILKOVI~, ANTONIO TORSELLO, TOSHIHIKO IIDA, EDUARDO ROJAS a n d KEVlN J. CATT* Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, U.S.A.
Summary--In cultured pituitary gonadotrophs, gonadotropin-releasing hormone (GnRH) caused dose-dependent and biphasic increases in cytoplasmic calcium concentration ([Ca :+ ]~) and LH release. Both extra- and intracellular calcium pools participate in GnRH-induced elevation of [Ca:+ ]i and LH secretion. The spike phase of the [Ca2÷]i response represents the primary signal derived predominantly from the rapid mobilization of intracellular Ca 2÷. In contrast, the prolonged phase of the Ca 2+ signal depends exclusively on Ca 2÷ entry from the extracellular pool. The influx of Ca 2÷ occurs partially through dihydropyridine-sensitive calcium channels. Both [Ca~+]i and LH responses to increasing concentrations of GnRH occur over very similar time scales, suggesting that increasing degrees of receptor occupancy are transduced into amplitude-modulated Ca 2÷ responses, which in turn activate exocytosis in a linear manner. However, several lines of evidence indicated the complexity over the relationship between Ca: ÷ signaling and LH exocytosis. In contrast to [Ca2÷]i measurements in cell suspension, single cell Ca 2+ measurements revealed the existence of a more complicated pattern of Ca 2÷ response to GnRH, with a biphasic response to high agonist doses and prominent oscillatory responses to lower GnRH concentrations, with a log-linear correlation between GnRH dose and the frequency of Ca 2÷ spiking. In addition, analysis of the magnitudes of the [Ca2+]i and LH responses of gonadotrophs to a wide range of GnRH concentrations in the presence and absence of extracellular Ca:+, and to K ÷ and phorboi ester stimulation, showed non-linearity between these parameters with amplification of [Ca~+]i-mediated exocytosis. Studies on cell depleted of protein kinase C under conditions that did not change the LH pool suggested the participation of protein kinase C in this amplification, especially during the plateau phase of the secretory response to GnRH.
cium mobilization from intracellular stores [9]. This phase of the Ca 2÷ response correlates temporally with an early rise in Ins(1,4,5)P3 [1]. However, the capacity of this calcium pool is limited, and sustained agonist stimulation requires the entry of calcium across the plasma membrane to maintain the less prominent, but functionally important, plateau of elevated cytoplasmic calcium. While it is generally accepted that an Ins(1,4,5)P3 -dependent process is responsible for the spike phase, several mechanisms have been suggested to explain the Ca 2÷ influx and hormone release during the plateau phase of the [Ca:÷]i response [4-8]. The LH secretory response to G n R H stimulation also occurs in a biphasic manner [5, 6, 7, 10]. Such biphasic [Ca:+]i and secretory responses have also been observed in other secretory cells, including pituitary corticotrophs, somatotrophs, lactotrophs and pituitary tumor cells [11-14]. During the initial phase, Ins(l,4,5)P3 induces a transient release of Ca :+ from an intracellular pool to generate the spike phase of the Ca 2+
INTRODUCTION
Gonadatropin-releasing hormone (GnRH) action in pituitary gonadotrophs is coupled to phospholipase C-induced hydrolysis of polyphosphoinositides, with production of the two second messengers, Ins(1,4,5)P3 [1] and diacylglycerol [2], and translocation of protein kinase C to the plasma membrane [3]. The rise in cytoplasmic free calcium concentrations ([CaS÷]i) in cell suspensions during agonist stimulation under physiological conditions follow a characteristic biphasic pattern, with an early spike phase and a subsequent plateau phase [4-8]. The initial rapid increase in [CaS÷]i does not require the presence of extracellular calcium [4], indicating that Ca 2+ is derived primarily by cal-
Proceedings of the lOth International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991. *To whom correspondence should be addressed. 453
454
STANKO S. STOJILKOVI(~et al.
signal. It has been proposed that this brief increase in [Ca2+]i induces only transient activation of calmodulin-dependent enzymes and that the sustained phase (with [Ca2+]~ slightly above the basal level) is maintained by protein kinase C. Although it has been useful to divide the cellular response into two distinct phases with two different intraceUular signals that are responsible for the initiation and the maintenance of the secretory response, it has become evident that the process is more complex and may require the synergistic action of calmodulin kinases and protein kinase C during the plateau phase of secretion [15]. Similarly, the synergistic interaction between calmodulin kinase II and protein kinase C was found to be a crucial factor for long-term potentiation of synaptic transmission[16], a physiological response of hippocampal neurons that is analogous to the sustained secretory response of endocrine cells. Our previous [7, 17] and present data also show the complexity of the interactions between the cellular events that occur during the biphasic [Ca2+]~ and secretory responses of gonadotrophs. The combination of frequent LH sampling with accurate measurements of [Ca 2+]i and of calcium current has provided a detailed and informative analysis of the kinetics of the [Ca 2+]~ and secretory responses to GnRH. The specificity and temporal properties of the two responses were defined by studies that include biochemical, electrophysiological and pharmacological characterization.
EXPERIMENTAL
Materials Trypsin, trypsin inhibitor, nifedipine and DNAase were obtained from Sigma (St Louis, MO). GnRH was from Peninsula Labs (Belmont, CA). Fura-2 Am was purchased from Calbiochem (San Diego, CA) and stored at 20°C as a 1 mM solution in dimethylsulfoxide (Sigma). Bovine serum albumin (BSA) was obtained from Miles Labs (Elkhart, IN) and Cytodex-1 beads from Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Bay K 8644 was a gift of Dr A. Scriabine (Miles Labs, West Haven, CT). Culture medium: Earle's M199 (Gibco, Grand Island, NY) with 10% horse serum, 2.2 g/l NaHCO3 and antibiotics. Perfusion medium: Hanks' M199 (Gibco) with 25 mM Hepes, 0.1% BSA and antibiotics. Calciumdeficient perfusion medium: M 199 (NIH Media
and Tissue Unit) with total [Ca2+]e=5/~M as measured by Ca 2+ electrode. Extracellular medium for patch-clamp studies: 140 mM choline chloride, 1.3 mM MgC12, 5.2 mM CaC12, 10 mM sodium Hepes, pH 7.4. Pipette medium for whole-cell recording: 75 mM cesium glutamate, 75 mM CsC1, 2.5 mM magnesium ATP, 10 mM sodium Hepes, 0.1 mM sodium EGTA, pH 7.2. Elutriation medium: Hanks' M199 with 25 mM Hepes, 1% BSA without Ca 2+ or Mg 2+. Fura-2 and Indo-I assay buffer: Hanks' salt solution with 25 mM Hepes, 0.01% BSA, without phenol red.
Pituitary cell preparation and purification Experiments were performed on anterior pituitary cells from normal or ovariectomized adult female Sprague-Dawley rats (200-250 g) obtained from Charles River, Inc (Wilmington, MA). The rats were killed by decapitation and the pituitary glands were immediately removed and washed in M199 containing 0.3% BSA. Isolated anterior pituitary cells were prepared by trypsin digestion and physical dispersion [18]. Cell viability, as ascertained by trypan blue exclusion, was about 90%. Cells were then resuspended in culture medium or in corresponding M 199 for centrifugal elutriation, which was performed as described previously[19]. This method yielded eight cell fractions of which the sixth and seventh show a 4- to 8-fold enrichment of gonadotrophs from the initial cell suspension. Further purification of gonadotrophs from castrated animals were performed by discontinuous centrifugation in Percol [20]. These highly purified cells were used for measurement of Ca 2+ currents in patch clamp experiments.
Perfusion system For perifusion studies, the cells were incubated at 37°C with preswollen Cytodex-1 beads in culture medium under 5% CO2 and 95% air. The cells were kept in 50ml Falcon tubes overnight, and in 50-mm Petri dishes for the next 2 days. The beads were then resuspended in perifusion medium and loaded into a 500-ml perifusion chamber (Endotronics, Minneapolis, MN), 15-20 x 106 cells/chamber. The cells were perifused with the same medium for 120min, and then with appropriate medium for each experiment. Fractions were collected every minute or every 5 s, and stored at -20°C prior to radioimmunoassay using the RP-3 rat LH standard provided by the National Pituitary Agency (Baltimore, MD).
Mechanisms of GnRH action in pituitarygonadotrophs
Cytoplasmic calcium measurements Ca 2÷ measurements in cell suspensions were performed on cells loaded with Fura-2 AM for 30 min at 37°C. Two million cells were used for [Ca2+]i assay by fluorescence analysis (dual excitation wavelength; 340 and 390 nm in a 3-ml cuvette at 32°C in a Delta Scan Spectrofluorimeter (Photon Technology Inter. Inc.). [Ca2÷]~ values were calculated after correction of emission data collected at 500 nm for dye leakage and autofluorescence, as described by Grynkiewicz et al. [21]. For single cell calcium measurements, purified gonadotrophs plated on 25-mm cover slips coated with poly-L-lysine and loaded with 2/IM Indo-1 AM for 60min at 37°C were mounted at 24°C on the stage of an inverted Diaphot microscope attached to an intracellular calcium analysis system (Nikon, Inc., Melville, NY). Measurement of LH release by reverse hemolytic plaque assay confirmed that about 90% of the purified cells were gonadotrophs [22]. In terms of Ca 2+ responses to GnRH, identified gonadotrophs from castrated females were more sensitive and responded more uniformly than those from normal females. To avoid desensitization and inactivation of VSCC by high doses of GnRh [23], the K÷-stimulated cells were identified by their calcium responses to transient stimulation with 10-12M GnRH. All [Ca2+]i values were derived from a standard curve that was constructed by addition of known concentration of Ca 2+ to 10/~M Indo-I [24].
Immunoblot analysis of protein kinase C Cells (10 × 106/tube; 5 × 105 cells/ml) were incubated at 24°C with or without phorbol esters (Sigma) in suspension culture under various experimental paradigms. Following incubation, cells were washed twice with Ca2+/Mg 2÷- free phosphate buffer, 0.5 mM phenylmethylsulfonyl fluoride and 2 pg/ml leupetin, and then ruptured by sonication for 1 min in 200 ml of 20 mM Tris-C1 (containing 1 mM dithiotreitol, 2 mM EDTA, 5mM EGTA, 10% glycerol 0.5mM PMSF and 2 #g/ml leupetin). Cytosol and membrane fractions were prepared by centrifugation (100,000g for 1 h) and analyzed by sodium dodecyl sulfate-electrophoresis and immunoblotting as described previously [17], followed by autoradiography. Polyclonal goat antibody to brain protein kinase C was donated by Dr K. P. Huang, Section on Metabolic Regulation, ERRB, NIH. Bands corresponding to protein kinase C were quantified by densitom-
455
etry and expressed as percent of the appropriate control.
Electrophysiological measurements The whole-cell mode of the patch-clamp technique was used [20]. Pipettes were prepared from micro-hematocrit capillary tubes and had an open tip resistance in physiological saline in the range of 2.5 to 5 MfL All experiments were carried out at room temperature. After sealing to the surface of the cell (resistance > 20 G~), the membrane patch was broken by suction and the current transients were recorded under voltage-clamp conditions using a List EPC-7 amplifier (List Electronics, Darmstadt-Eberstsdt, Fed. Rep. Germany). The current records were filtered by means of an 8-pole 902LPF Bessel filter (Frequency Devices, Haverhill, MA) set at 8 kHz and then stored on-line on a computer (System 90, Computer Instrumentation Limited, Southampton, U.K.) for later analyses.
Calculations EDs0 values were calculated from logit-log plots. The statistical significance of linear relationships between x and y was evaluated by the correlation coefficient, r, used with (n-2) degrees of freedom and P < 0.001. The significances of differences between means were evaluated by Student's t test.
RESULTS AND DISCUSSION
Biphasic cytoplasmic free calcium and LH responses during agonist stimulation The availability of rapid assays of intracellular signals, including analysis of Ins(1,4,5)P3 production in [3H]inositol-labeled cells [1], Fura-2 measurements of [Ca2+]i in cell suspensions (1 s resolution time; Ref. [6]) or Indo-1 in single cells (0.3 s; Ref. [24]) and electrophysiological measurements (measured in ms; Ref. [20]) required the development of a comparable procedure for rapid resolution of the LH secretory profile during agonist stimulation. However, most studies on gonadotropin release have been performed in static cultures with assay of medium samples every 30 to 60 min for 2 to 4 h. This approach does not permit a valid comparison between the signaling and secretion responses to agonist stimulation. We [6] and others [10] have employed perifusion of pituitary cells, with collection of effluent every 1 to 5 min, in the analysis of temporal changes in LH
STANKOS. STOJILKOVI~et al.
456
secretion during pulsatile exposure to GnRH under various experimental conditions. This system was further developed to allow the comparison between signaling and secretion, with collection of samples every 5 s. The GnRH concentration profile and washout time in the perifusion column were estimated 150
with [125I]-GnRH. The temporal changes in [t25I]-GnRH concentrations in the perifusion chamber during the GnRH pulse followed by perifusion medium are presented in Fig. I(A). The GnRH profile showed an exponential rise with a half-time of 45 s, that reached the maximum value of 100% of the original [125I]-GnRH concentration after 2.4 min., and was followed by an exponential fall during the washing period. The time required to wash out 50% of
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Fig I. BiphasJc L H response and comparison with profile of stimulatory O n R H pulse during cellcolumn perifusion. (A) G n R H concentrationand wash-out time in perifusion columns. Temporal changes in G n R H concentrationswere estimated by infusion of [t2Sl]-OnRH [40], with 5 s collection of fractions. (B) Profile of LH secretion during cell column perifusion with normal and calcium deficient medium. Perifusion with 5 #M Ca 2+ reduced the spike phase by 20-40% and almost abolished the second phase (the residual LH release during the plateau phase represents 15-20% of the control). (C) Recovery of the ¢xtracellular Ca2+-dependent part of LH release during GnRH stimulation. A GnRH pulse was applied under extracellular Ca2+-deficient conditions ([Ca2+],=5vM) and 1.25mM Ca 2+ was restored 4min after the beginning of the stimulation, as indicated by arrow. Upper curve, biphasic LH response during the control pulse of GnRH.
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T i me (seconds) Fig. 2. Participation of extracellular calcium in the [Ca2+]i response to GnRH. (A) Biphasic [Ca2+]i response to 100 nM GnRH. Upper curve, [Ca:+], = 1.25 mM; lower curve, [Ca~+]i = 5 ,a M. The tracing represent the computer-derived means of data from five independent experiments. The calcium curve obtained under Ca2+-deficient conditions is significantly different from controls at all points during GnRH action (P < 0.05). (B, and C) Extracellular Ca2+-dependence of GnRH-induced cytoplasmic calcium responses in single gonadotrophs. +Ca 2+ = 1.25 mM; - C a ~+ = 200 riM.
Mechanisms of GnRH action in pituitary gonadotrophs the maximum []:5I]-GnRH concentration in the column was 35 s. This type of G n R H pulse induced a characteristic biphasic pattern of changes in L H release, with an acute spike followed by a prolonged plateau phase. As shown in Fig. I(B), upper curve, measurable gonadotropin release was observed 5 s after exposure to the agonist; the peak in gonadotropin release occurred at 35 s and the total duration of the initial phase of L H release was 2.5 min. In the presence of extracellular Ca 2÷, the plateau phase remained stable during the first 15 min of perifusion at 30 to 50% of the peak value of the L H response. The tail secretion of L H showed an exponential decline, with a half-time of 1.2 min. The spike phase of the LH response was reduced in magnitude and the plateau phase was strongly attenuated in extracellular CaS+-deficient medium [ 5 # M Ca :+, Fig. I(B), lower curve]• On the other hand, recovery of the extracellular Ca :+dependent component of L H release during G n R H stimulation was a rapid process, as indicated in Fig. I(C), and occurred within 20 to 30 s, with a temporal overshooting, The characteristic peak and plateau phases of LH release were accompanied by similar changes in the [Ca 2÷]~profile of pituitary cells in suspension during agonist stimulation [Fig. 2(A), upper curve]. The maximum rise in [CaS+]i occurred within 20 s, and the total duration of the spike phase was about 2.2 min. The magnitude of the subsequent phase did not exceed about 20% of the maximum response during the spike phase• In CaS÷-deficient medium, the magnitude of the acute phase of the [Ca2+]i response was reduced by 20 to 40% and the second phase was completely abolished [Fig. 2(A), bottom curve]. The inability of secretagogues to mobilize the residual calcium from Ca2+-deficient medium was supported by the absence of [Ca2+]i and secretory responses to depolarization by K÷; the basal levels of [Ca:+]i and L H release were 122 + 7 nM and 5.2 + 0.4 ng/ml; the levels of [CaS+]~ and L H release in 50 mM K ÷ in Ca 2÷deficient medium was 128 + 6 nM and 5.4 +__0.5 ng/ml; and in 5 0 m M K ÷ in the presence of 1.25 mM Ca 2+ were 428 + 19 nM and 41.1 + 2.5 ng/ml. Similar effects of depletion of extracellular calcium on Ca 2+ signaling were observed in single gonadotrophs stimulated with 100nM GnRH. As shown in Fig. 2(C), after removal of extracellular Ca 2+ from the medium the G n R H evoked biphasic responses in [Ca:+]~ declined to
457
baseline after 2-3 min. In contrast, the plateau phase persisted in Ca:+-containing medium for at least 10 min [Fig. 2(B)], in good agreement with results obtained in pituitary cell suspensions [Fig. 2(A)]. 10 rain r
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Fig. 3. Effectsof the calciumchannel antagonists, cadmium, cobalt and nifedipine, on GnRH-induced LH release. (A) CdCl2 (100/JM) was added in calcium-containingmedium and applied 3 win before, during and 5 win after the GnRH pulse. The values shown w e r e obtained from fractions collected for 1 min. (B) Dose-dependenteffectsof Co2+ on GnRH- induced LH release. Experiments were obtained from the same columnduring repetitive(10 min) stimulation by C-nRH (10nM, 2h-t). CoCl2 was added in Ca ~+containing medium and applied 3 min before, during and 5 win after GnRH pulses. The values shown were obtained during I win collections. (C) The dihydropyridinecalcium channel antagonist, nifedipine (l/zM) was added 5min before, during and 5 min after the GnRH pulse. Samples from the perifusion column were collected every 5s.
458
STANKO S. STOJILKOVI6 et al.
These data have confirmed that a rapid increase in [Ca2+]i is the primary signal leading to LH secretion and is predominantly generated by mobilization of intracellular Ca 2+. The kinetics of Ins(1,4,5)P 3 formation during GnRH stimulation[l] are in agreement with these conclusions. On the other hand, Ca 2+ entry is the dominant source of Ca 2+ during the plateau phase but also contributes significantly during the spike phase. These findings are not in accord with the generally accepted model of the two phases and two source compartments of Ca 2+, and indicate that the two temporal phases of the [ C a 2 + ]i and LH response do not bear a simple one-to-one relationship with the two source components of calcium, since Ca 2+ influx already participates in the spike phase.
Characterization of the extracellular Ca 2+dependent mechanism of GnRH action The non-specific calcium channel antagonist, Cd 2+, completely inhibited the extracellular-calcium-dependent component of GnRH-induced LH release at 100 pM doses [Fig. 3(A)]. However, the secretion of LH was almost abolished during repetitive stimulation with GnRH (10 nM) in the presence of the same dose of C d 2+, indicating the toxicity of the blocker in long-term studies. Another non-specific calcium channel blocker, Co 2+, also reduced LH secretion during agonist stimulation in a dosedependent manner. Similar to Cd 2+, comparison between the effects of Co 2+ and the effects of Ca 2+ depletion on GnRH-induced gonadotropin release suggests that Co 2÷ not only causes blockade of Ca 2+ entry but also attenuates the extracellular Ca2÷-independent component of LH secretion. As shown in Fig. 3(B), the inhibitory effects of 0.5, 1.0 and 2.5 mM Co 2+ on both phases of LH response exceeded those caused by depletion of extracellular Ca 2+ alone. Such an action of Co 2+ at an intracellular site is not surprising because this ion, in addition to blocking calcium current, can enter the cytoplasm of GH3 cells and inhibit prolactin secretion in the absence of Ca 2+ influx [25]. On the other hand, the dihydropyridine calcium channel antagonist, nifedipine, had only a partial inhibitory effect on both spike and plateau phases of the LH response [Fig. 3(C)]; the extracellular-Ca2+-dependent component of secretion was reduced by 45 to 55% in the presence of 5 #M nifedipine. In contrast, the dihydropyridine calcium channel agonist, Bay K 8644, caused dose-dependent elevation of
[C a 2 + ]i and LH response and markedly enhanced KCl-induced elevations of [Ca2+]i and LH release. As shown in Fig. 4(B), Bay K 8644 caused dose-dependent enhancement of LH release, with increases in both the sensitivity and magnitude of the response as the KC1 concentration was raised from 5 to 30 mM. In the same cell preparation, measurement of [Ca2+]i revealed that Bay K 8644 caused dose-dependent elevation of [Ca2+]i and markedly enhanced KCl-induced elevation of [Ca2+]i [Fig. 4(A)]. Such data are indicative of the presence and participation of VSCC in pituitary gonadotrophs, and this has been further confirmed by patch-clamp experiments. Figure 5(A) shows a set of superimposed membrane current records from a pituitary gonadotroph internally dialyzed with K+-free Cs + solution and bathed in a modified Krebs buffer. Figure 5(B) shows the current-voltage relationship (I-V curves) for peak and steady-state inward current with Ca 2+.
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CALCIUM
SIGNALING
AGONIST-STIMULATED
AND
0960-0760/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press plc
SECRETORY
PITUITARY
RESPONSES
IN
GONADOTROPHS
STANKO S. STOJILKOVI~, ANTONIO TORSELLO, TOSHIHIKO IIDA, EDUARDO ROJAS a n d KEVlN J. CATT* Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, U.S.A.
Summary--In cultured pituitary gonadotrophs, gonadotropin-releasing hormone (GnRH) caused dose-dependent and biphasic increases in cytoplasmic calcium concentration ([Ca :+ ]~) and LH release. Both extra- and intracellular calcium pools participate in GnRH-induced elevation of [Ca:+ ]i and LH secretion. The spike phase of the [Ca2÷]i response represents the primary signal derived predominantly from the rapid mobilization of intracellular Ca 2÷. In contrast, the prolonged phase of the Ca 2+ signal depends exclusively on Ca 2÷ entry from the extracellular pool. The influx of Ca 2÷ occurs partially through dihydropyridine-sensitive calcium channels. Both [Ca~+]i and LH responses to increasing concentrations of GnRH occur over very similar time scales, suggesting that increasing degrees of receptor occupancy are transduced into amplitude-modulated Ca 2÷ responses, which in turn activate exocytosis in a linear manner. However, several lines of evidence indicated the complexity over the relationship between Ca: ÷ signaling and LH exocytosis. In contrast to [Ca2÷]i measurements in cell suspension, single cell Ca 2+ measurements revealed the existence of a more complicated pattern of Ca 2÷ response to GnRH, with a biphasic response to high agonist doses and prominent oscillatory responses to lower GnRH concentrations, with a log-linear correlation between GnRH dose and the frequency of Ca 2÷ spiking. In addition, analysis of the magnitudes of the [Ca2+]i and LH responses of gonadotrophs to a wide range of GnRH concentrations in the presence and absence of extracellular Ca:+, and to K ÷ and phorboi ester stimulation, showed non-linearity between these parameters with amplification of [Ca~+]i-mediated exocytosis. Studies on cell depleted of protein kinase C under conditions that did not change the LH pool suggested the participation of protein kinase C in this amplification, especially during the plateau phase of the secretory response to GnRH.
cium mobilization from intracellular stores [9]. This phase of the Ca 2÷ response correlates temporally with an early rise in Ins(1,4,5)P3 [1]. However, the capacity of this calcium pool is limited, and sustained agonist stimulation requires the entry of calcium across the plasma membrane to maintain the less prominent, but functionally important, plateau of elevated cytoplasmic calcium. While it is generally accepted that an Ins(1,4,5)P3 -dependent process is responsible for the spike phase, several mechanisms have been suggested to explain the Ca 2÷ influx and hormone release during the plateau phase of the [Ca:÷]i response [4-8]. The LH secretory response to G n R H stimulation also occurs in a biphasic manner [5, 6, 7, 10]. Such biphasic [Ca:+]i and secretory responses have also been observed in other secretory cells, including pituitary corticotrophs, somatotrophs, lactotrophs and pituitary tumor cells [11-14]. During the initial phase, Ins(l,4,5)P3 induces a transient release of Ca :+ from an intracellular pool to generate the spike phase of the Ca 2+
INTRODUCTION
Gonadatropin-releasing hormone (GnRH) action in pituitary gonadotrophs is coupled to phospholipase C-induced hydrolysis of polyphosphoinositides, with production of the two second messengers, Ins(1,4,5)P3 [1] and diacylglycerol [2], and translocation of protein kinase C to the plasma membrane [3]. The rise in cytoplasmic free calcium concentrations ([CaS÷]i) in cell suspensions during agonist stimulation under physiological conditions follow a characteristic biphasic pattern, with an early spike phase and a subsequent plateau phase [4-8]. The initial rapid increase in [CaS÷]i does not require the presence of extracellular calcium [4], indicating that Ca 2+ is derived primarily by cal-
Proceedings of the lOth International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991. *To whom correspondence should be addressed. 453
454
STANKO S. STOJILKOVI(~et al.
signal. It has been proposed that this brief increase in [Ca2+]i induces only transient activation of calmodulin-dependent enzymes and that the sustained phase (with [Ca2+]~ slightly above the basal level) is maintained by protein kinase C. Although it has been useful to divide the cellular response into two distinct phases with two different intraceUular signals that are responsible for the initiation and the maintenance of the secretory response, it has become evident that the process is more complex and may require the synergistic action of calmodulin kinases and protein kinase C during the plateau phase of secretion [15]. Similarly, the synergistic interaction between calmodulin kinase II and protein kinase C was found to be a crucial factor for long-term potentiation of synaptic transmission[16], a physiological response of hippocampal neurons that is analogous to the sustained secretory response of endocrine cells. Our previous [7, 17] and present data also show the complexity of the interactions between the cellular events that occur during the biphasic [Ca2+]~ and secretory responses of gonadotrophs. The combination of frequent LH sampling with accurate measurements of [Ca 2+]i and of calcium current has provided a detailed and informative analysis of the kinetics of the [Ca 2+]~ and secretory responses to GnRH. The specificity and temporal properties of the two responses were defined by studies that include biochemical, electrophysiological and pharmacological characterization.
EXPERIMENTAL
Materials Trypsin, trypsin inhibitor, nifedipine and DNAase were obtained from Sigma (St Louis, MO). GnRH was from Peninsula Labs (Belmont, CA). Fura-2 Am was purchased from Calbiochem (San Diego, CA) and stored at 20°C as a 1 mM solution in dimethylsulfoxide (Sigma). Bovine serum albumin (BSA) was obtained from Miles Labs (Elkhart, IN) and Cytodex-1 beads from Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Bay K 8644 was a gift of Dr A. Scriabine (Miles Labs, West Haven, CT). Culture medium: Earle's M199 (Gibco, Grand Island, NY) with 10% horse serum, 2.2 g/l NaHCO3 and antibiotics. Perfusion medium: Hanks' M199 (Gibco) with 25 mM Hepes, 0.1% BSA and antibiotics. Calciumdeficient perfusion medium: M 199 (NIH Media
and Tissue Unit) with total [Ca2+]e=5/~M as measured by Ca 2+ electrode. Extracellular medium for patch-clamp studies: 140 mM choline chloride, 1.3 mM MgC12, 5.2 mM CaC12, 10 mM sodium Hepes, pH 7.4. Pipette medium for whole-cell recording: 75 mM cesium glutamate, 75 mM CsC1, 2.5 mM magnesium ATP, 10 mM sodium Hepes, 0.1 mM sodium EGTA, pH 7.2. Elutriation medium: Hanks' M199 with 25 mM Hepes, 1% BSA without Ca 2+ or Mg 2+. Fura-2 and Indo-I assay buffer: Hanks' salt solution with 25 mM Hepes, 0.01% BSA, without phenol red.
Pituitary cell preparation and purification Experiments were performed on anterior pituitary cells from normal or ovariectomized adult female Sprague-Dawley rats (200-250 g) obtained from Charles River, Inc (Wilmington, MA). The rats were killed by decapitation and the pituitary glands were immediately removed and washed in M199 containing 0.3% BSA. Isolated anterior pituitary cells were prepared by trypsin digestion and physical dispersion [18]. Cell viability, as ascertained by trypan blue exclusion, was about 90%. Cells were then resuspended in culture medium or in corresponding M 199 for centrifugal elutriation, which was performed as described previously[19]. This method yielded eight cell fractions of which the sixth and seventh show a 4- to 8-fold enrichment of gonadotrophs from the initial cell suspension. Further purification of gonadotrophs from castrated animals were performed by discontinuous centrifugation in Percol [20]. These highly purified cells were used for measurement of Ca 2+ currents in patch clamp experiments.
Perfusion system For perifusion studies, the cells were incubated at 37°C with preswollen Cytodex-1 beads in culture medium under 5% CO2 and 95% air. The cells were kept in 50ml Falcon tubes overnight, and in 50-mm Petri dishes for the next 2 days. The beads were then resuspended in perifusion medium and loaded into a 500-ml perifusion chamber (Endotronics, Minneapolis, MN), 15-20 x 106 cells/chamber. The cells were perifused with the same medium for 120min, and then with appropriate medium for each experiment. Fractions were collected every minute or every 5 s, and stored at -20°C prior to radioimmunoassay using the RP-3 rat LH standard provided by the National Pituitary Agency (Baltimore, MD).
Mechanisms of GnRH action in pituitarygonadotrophs
Cytoplasmic calcium measurements Ca 2÷ measurements in cell suspensions were performed on cells loaded with Fura-2 AM for 30 min at 37°C. Two million cells were used for [Ca2+]i assay by fluorescence analysis (dual excitation wavelength; 340 and 390 nm in a 3-ml cuvette at 32°C in a Delta Scan Spectrofluorimeter (Photon Technology Inter. Inc.). [Ca2÷]~ values were calculated after correction of emission data collected at 500 nm for dye leakage and autofluorescence, as described by Grynkiewicz et al. [21]. For single cell calcium measurements, purified gonadotrophs plated on 25-mm cover slips coated with poly-L-lysine and loaded with 2/IM Indo-1 AM for 60min at 37°C were mounted at 24°C on the stage of an inverted Diaphot microscope attached to an intracellular calcium analysis system (Nikon, Inc., Melville, NY). Measurement of LH release by reverse hemolytic plaque assay confirmed that about 90% of the purified cells were gonadotrophs [22]. In terms of Ca 2+ responses to GnRH, identified gonadotrophs from castrated females were more sensitive and responded more uniformly than those from normal females. To avoid desensitization and inactivation of VSCC by high doses of GnRh [23], the K÷-stimulated cells were identified by their calcium responses to transient stimulation with 10-12M GnRH. All [Ca2+]i values were derived from a standard curve that was constructed by addition of known concentration of Ca 2+ to 10/~M Indo-I [24].
Immunoblot analysis of protein kinase C Cells (10 × 106/tube; 5 × 105 cells/ml) were incubated at 24°C with or without phorbol esters (Sigma) in suspension culture under various experimental paradigms. Following incubation, cells were washed twice with Ca2+/Mg 2÷- free phosphate buffer, 0.5 mM phenylmethylsulfonyl fluoride and 2 pg/ml leupetin, and then ruptured by sonication for 1 min in 200 ml of 20 mM Tris-C1 (containing 1 mM dithiotreitol, 2 mM EDTA, 5mM EGTA, 10% glycerol 0.5mM PMSF and 2 #g/ml leupetin). Cytosol and membrane fractions were prepared by centrifugation (100,000g for 1 h) and analyzed by sodium dodecyl sulfate-electrophoresis and immunoblotting as described previously [17], followed by autoradiography. Polyclonal goat antibody to brain protein kinase C was donated by Dr K. P. Huang, Section on Metabolic Regulation, ERRB, NIH. Bands corresponding to protein kinase C were quantified by densitom-
455
etry and expressed as percent of the appropriate control.
Electrophysiological measurements The whole-cell mode of the patch-clamp technique was used [20]. Pipettes were prepared from micro-hematocrit capillary tubes and had an open tip resistance in physiological saline in the range of 2.5 to 5 MfL All experiments were carried out at room temperature. After sealing to the surface of the cell (resistance > 20 G~), the membrane patch was broken by suction and the current transients were recorded under voltage-clamp conditions using a List EPC-7 amplifier (List Electronics, Darmstadt-Eberstsdt, Fed. Rep. Germany). The current records were filtered by means of an 8-pole 902LPF Bessel filter (Frequency Devices, Haverhill, MA) set at 8 kHz and then stored on-line on a computer (System 90, Computer Instrumentation Limited, Southampton, U.K.) for later analyses.
Calculations EDs0 values were calculated from logit-log plots. The statistical significance of linear relationships between x and y was evaluated by the correlation coefficient, r, used with (n-2) degrees of freedom and P < 0.001. The significances of differences between means were evaluated by Student's t test.
RESULTS AND DISCUSSION
Biphasic cytoplasmic free calcium and LH responses during agonist stimulation The availability of rapid assays of intracellular signals, including analysis of Ins(1,4,5)P3 production in [3H]inositol-labeled cells [1], Fura-2 measurements of [Ca2+]i in cell suspensions (1 s resolution time; Ref. [6]) or Indo-1 in single cells (0.3 s; Ref. [24]) and electrophysiological measurements (measured in ms; Ref. [20]) required the development of a comparable procedure for rapid resolution of the LH secretory profile during agonist stimulation. However, most studies on gonadotropin release have been performed in static cultures with assay of medium samples every 30 to 60 min for 2 to 4 h. This approach does not permit a valid comparison between the signaling and secretion responses to agonist stimulation. We [6] and others [10] have employed perifusion of pituitary cells, with collection of effluent every 1 to 5 min, in the analysis of temporal changes in LH
STANKOS. STOJILKOVI~et al.
456
secretion during pulsatile exposure to GnRH under various experimental conditions. This system was further developed to allow the comparison between signaling and secretion, with collection of samples every 5 s. The GnRH concentration profile and washout time in the perifusion column were estimated 150
with [125I]-GnRH. The temporal changes in [t25I]-GnRH concentrations in the perifusion chamber during the GnRH pulse followed by perifusion medium are presented in Fig. I(A). The GnRH profile showed an exponential rise with a half-time of 45 s, that reached the maximum value of 100% of the original [125I]-GnRH concentration after 2.4 min., and was followed by an exponential fall during the washing period. The time required to wash out 50% of
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Fig I. BiphasJc L H response and comparison with profile of stimulatory O n R H pulse during cellcolumn perifusion. (A) G n R H concentrationand wash-out time in perifusion columns. Temporal changes in G n R H concentrationswere estimated by infusion of [t2Sl]-OnRH [40], with 5 s collection of fractions. (B) Profile of LH secretion during cell column perifusion with normal and calcium deficient medium. Perifusion with 5 #M Ca 2+ reduced the spike phase by 20-40% and almost abolished the second phase (the residual LH release during the plateau phase represents 15-20% of the control). (C) Recovery of the ¢xtracellular Ca2+-dependent part of LH release during GnRH stimulation. A GnRH pulse was applied under extracellular Ca2+-deficient conditions ([Ca2+],=5vM) and 1.25mM Ca 2+ was restored 4min after the beginning of the stimulation, as indicated by arrow. Upper curve, biphasic LH response during the control pulse of GnRH.
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T i me (seconds) Fig. 2. Participation of extracellular calcium in the [Ca2+]i response to GnRH. (A) Biphasic [Ca2+]i response to 100 nM GnRH. Upper curve, [Ca:+], = 1.25 mM; lower curve, [Ca~+]i = 5 ,a M. The tracing represent the computer-derived means of data from five independent experiments. The calcium curve obtained under Ca2+-deficient conditions is significantly different from controls at all points during GnRH action (P < 0.05). (B, and C) Extracellular Ca2+-dependence of GnRH-induced cytoplasmic calcium responses in single gonadotrophs. +Ca 2+ = 1.25 mM; - C a ~+ = 200 riM.
Mechanisms of GnRH action in pituitary gonadotrophs the maximum []:5I]-GnRH concentration in the column was 35 s. This type of G n R H pulse induced a characteristic biphasic pattern of changes in L H release, with an acute spike followed by a prolonged plateau phase. As shown in Fig. I(B), upper curve, measurable gonadotropin release was observed 5 s after exposure to the agonist; the peak in gonadotropin release occurred at 35 s and the total duration of the initial phase of L H release was 2.5 min. In the presence of extracellular Ca 2÷, the plateau phase remained stable during the first 15 min of perifusion at 30 to 50% of the peak value of the L H response. The tail secretion of L H showed an exponential decline, with a half-time of 1.2 min. The spike phase of the LH response was reduced in magnitude and the plateau phase was strongly attenuated in extracellular CaS+-deficient medium [ 5 # M Ca :+, Fig. I(B), lower curve]• On the other hand, recovery of the extracellular Ca :+dependent component of L H release during G n R H stimulation was a rapid process, as indicated in Fig. I(C), and occurred within 20 to 30 s, with a temporal overshooting, The characteristic peak and plateau phases of LH release were accompanied by similar changes in the [Ca 2÷]~profile of pituitary cells in suspension during agonist stimulation [Fig. 2(A), upper curve]. The maximum rise in [CaS+]i occurred within 20 s, and the total duration of the spike phase was about 2.2 min. The magnitude of the subsequent phase did not exceed about 20% of the maximum response during the spike phase• In CaS÷-deficient medium, the magnitude of the acute phase of the [Ca2+]i response was reduced by 20 to 40% and the second phase was completely abolished [Fig. 2(A), bottom curve]. The inability of secretagogues to mobilize the residual calcium from Ca2+-deficient medium was supported by the absence of [Ca2+]i and secretory responses to depolarization by K÷; the basal levels of [Ca:+]i and L H release were 122 + 7 nM and 5.2 + 0.4 ng/ml; the levels of [CaS+]~ and L H release in 50 mM K ÷ in Ca 2÷deficient medium was 128 + 6 nM and 5.4 +__0.5 ng/ml; and in 5 0 m M K ÷ in the presence of 1.25 mM Ca 2+ were 428 + 19 nM and 41.1 + 2.5 ng/ml. Similar effects of depletion of extracellular calcium on Ca 2+ signaling were observed in single gonadotrophs stimulated with 100nM GnRH. As shown in Fig. 2(C), after removal of extracellular Ca 2+ from the medium the G n R H evoked biphasic responses in [Ca:+]~ declined to
457
baseline after 2-3 min. In contrast, the plateau phase persisted in Ca:+-containing medium for at least 10 min [Fig. 2(B)], in good agreement with results obtained in pituitary cell suspensions [Fig. 2(A)]. 10 rain r
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Fig. 3. Effectsof the calciumchannel antagonists, cadmium, cobalt and nifedipine, on GnRH-induced LH release. (A) CdCl2 (100/JM) was added in calcium-containingmedium and applied 3 win before, during and 5 win after the GnRH pulse. The values shown w e r e obtained from fractions collected for 1 min. (B) Dose-dependenteffectsof Co2+ on GnRH- induced LH release. Experiments were obtained from the same columnduring repetitive(10 min) stimulation by C-nRH (10nM, 2h-t). CoCl2 was added in Ca ~+containing medium and applied 3 min before, during and 5 win after GnRH pulses. The values shown were obtained during I win collections. (C) The dihydropyridinecalcium channel antagonist, nifedipine (l/zM) was added 5min before, during and 5 min after the GnRH pulse. Samples from the perifusion column were collected every 5s.
458
STANKO S. STOJILKOVI6 et al.
These data have confirmed that a rapid increase in [Ca2+]i is the primary signal leading to LH secretion and is predominantly generated by mobilization of intracellular Ca 2+. The kinetics of Ins(1,4,5)P 3 formation during GnRH stimulation[l] are in agreement with these conclusions. On the other hand, Ca 2+ entry is the dominant source of Ca 2+ during the plateau phase but also contributes significantly during the spike phase. These findings are not in accord with the generally accepted model of the two phases and two source compartments of Ca 2+, and indicate that the two temporal phases of the [ C a 2 + ]i and LH response do not bear a simple one-to-one relationship with the two source components of calcium, since Ca 2+ influx already participates in the spike phase.
Characterization of the extracellular Ca 2+dependent mechanism of GnRH action The non-specific calcium channel antagonist, Cd 2+, completely inhibited the extracellular-calcium-dependent component of GnRH-induced LH release at 100 pM doses [Fig. 3(A)]. However, the secretion of LH was almost abolished during repetitive stimulation with GnRH (10 nM) in the presence of the same dose of C d 2+, indicating the toxicity of the blocker in long-term studies. Another non-specific calcium channel blocker, Co 2+, also reduced LH secretion during agonist stimulation in a dosedependent manner. Similar to Cd 2+, comparison between the effects of Co 2+ and the effects of Ca 2+ depletion on GnRH-induced gonadotropin release suggests that Co 2÷ not only causes blockade of Ca 2+ entry but also attenuates the extracellular Ca2÷-independent component of LH secretion. As shown in Fig. 3(B), the inhibitory effects of 0.5, 1.0 and 2.5 mM Co 2+ on both phases of LH response exceeded those caused by depletion of extracellular Ca 2+ alone. Such an action of Co 2+ at an intracellular site is not surprising because this ion, in addition to blocking calcium current, can enter the cytoplasm of GH3 cells and inhibit prolactin secretion in the absence of Ca 2+ influx [25]. On the other hand, the dihydropyridine calcium channel antagonist, nifedipine, had only a partial inhibitory effect on both spike and plateau phases of the LH response [Fig. 3(C)]; the extracellular-Ca2+-dependent component of secretion was reduced by 45 to 55% in the presence of 5 #M nifedipine. In contrast, the dihydropyridine calcium channel agonist, Bay K 8644, caused dose-dependent elevation of
[C a 2 + ]i and LH response and markedly enhanced KCl-induced elevations of [Ca2+]i and LH release. As shown in Fig. 4(B), Bay K 8644 caused dose-dependent enhancement of LH release, with increases in both the sensitivity and magnitude of the response as the KC1 concentration was raised from 5 to 30 mM. In the same cell preparation, measurement of [Ca2+]i revealed that Bay K 8644 caused dose-dependent elevation of [Ca2+]i and markedly enhanced KCl-induced elevation of [Ca2+]i [Fig. 4(A)]. Such data are indicative of the presence and participation of VSCC in pituitary gonadotrophs, and this has been further confirmed by patch-clamp experiments. Figure 5(A) shows a set of superimposed membrane current records from a pituitary gonadotroph internally dialyzed with K+-free Cs + solution and bathed in a modified Krebs buffer. Figure 5(B) shows the current-voltage relationship (I-V curves) for peak and steady-state inward current with Ca 2+.
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