Mechanism of agonist-induced [Ca2+] i oscillations in pituitary ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Val. 268, No. 11, Issue of April 15, pp. 7713-7720,1993 Printed in U.S.A.

Mechanism of Agonist-induced [Ca2+IiOscillations in Pituitary Gonadotrophs” (Received for publication, July 23, 1992)

Stanko S. StojilkoviiS, Manuel Kukuljang, Melanija Tomii, Eduardo Rojasg, and KevinJ. Catt From the Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Developmentand the §Laboratoryof Cell Biology and Genetics, National Instituteof Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland20892

oscillations, and theirphysiological significance, are currently Gonadotropin-releasing hormone (GnRH) activates oscillatory Ca2+ signaling in pituitary gonadotrophs at the subjectsof intensive investigation (reviewed in Tsien and a frequency (up to 2 5 min”) that is dose-dependent Tsien, 1990). In general, such oscillations have been proposed and is determined by the degree of receptor-mediated to be either “receptor-controlled,’’ i.e. generated by fluctuating inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation. levels of inositol (1,4,5) trisphosphate (Ins(1,4,5)P3),’ or “secSimilar dose-dependent and frequency-modulated Ca2+ond-messenger-controlled,” i.e. occurring in the absence of oscillations were elicitedby intracellular administra- Ins(1,4,5)P3oscillations (Berridge, 1990).Based on these contion of Ins(1,4,5)P3 and its nonhydrolyzable analogs, cepts,andonthepotential positive or negative feedback consistent with models in which Ins(1,4,5)P3 levels effects of [Ca”], several theoretical schemes and their cordo not responding mathematical models havebeen developed (redetermine the frequencyof Ca2+ oscillations but fluctuate in synchrony with [Ca2+li.At constant agonist viewed in Stojilkovic and Catt, 1992). concentrations, Ca2+ spiking varied in amplitude, with a number of progressively larger transients before the Gonadotropin-releasing hormone (GnRH) belongs to the onset of maximal oscillations, followed by a gradual Ca2+-mobilizing groupof hormones, and its secretory actions decrease in spike amplitude that was accompanied by in gonadotrophsare associatedwith rapidproduction of Ins(1,4,5)P3 (Morgan et al., 1987) and diacylglycerol (Chang an increase in spiking frequency. The decline in the of Ca2+transients et al., 1988). These responses are accompanied by prominent amplitude and increase in frequency (Leong and Thorner, 1991; during stimulation by GnRH were not related to a oscillatory elevations in [Ca2++Ii decrease in the propagationof the Ca2+ signal within Stojilkovic et al., 1990; Shangold et al., 1988) that are highly the cell but were associated with gradual depletionof organized both temporally (Iida et al., 1991) and spatially the agonist-sensitive Ca2+pool. Once initiated, the pat- (Rawlings et al., 1991). Another Ca2+-mobilizingagonist, entern of Ca2+ spiking was not altered by blockade of dothelin, induces a similar pattern of Ca2+ responses in goreceptor occupancy, by inhibition of phospholipase C, nadotrophs, acting through a specific ETA receptor subtype or by reduction of extracellular [Ca”’]. Also, the en- (Stojilkovic et al., 1990, 1992a). The activation of GnRH or doplasmicreticulum(Ca2+)-ATPaseblocker,thapsiendothelin receptors in gonadotrophs by increasing agonist gargin, could substitute for Ins(1,4,5)P3 in initiating concentrations leads to four distinct steps in the single-cell the oscillatory Ca2+ response. These findings indicate [Ca’+]; response asfollows: (i) subthreshold,in which increasthat althoughtheIns(1,4,5)P3concentrationdetering agonist concentrations elicit Ca2+ responsesof increasing mines the patternof transients at the initiation of the amplitude; (ii) attainment of a threshold [Ca2+];level that of the signaldoes appears to initiate Ca2+ spiking; (iii) modulation of the freoscillatory Ca2+ signal, maintenance not require a sustained rise in Ins(1,4,5)P3. Since the quency but not the amplitude of Ca’+ transients by increasing frequency of Ca2+ oscillations is alsoinfluencedby agonist concentrations; (iv) “saturation” of the oscillatory depletion of luminal[Ca2+], it is possible thatthe Ins( 1,4,5)P3-sensitive channels in the endoplasmic re- response, leading to a biphasic (spike and plateau) response (Iida et al., 1991). ticulum are tonically inhibited by high intraluminal Ca2+ levelsand that Ins( 1,4,5)P3 surmounts such inhi-GnRH-induced Ca2+ transients can be elicited in voltagebition by promoting Ca2+ discharge. When a critical clamped cells and are independent of extracellular Ca’+, in in is attained, repetitiveCa2+tran- accord with the operation of a“cytoplasmicoscillator” level of Ca2+ discharge sients are generated by an autocatalyticmechanism in agonist-stimulated gonadotrophs (Stojilkovic et al., 1992b). which a sustained rise in Ins( 1,4,5)P3 is not an essential However, depletion of extracellular Ca2+ usually limits the requirement. duration of the oscillatory [Ca2+]; signal to 200-300 s (Iida et al., 1991). In the presence of extracellular calcium, low-amplitude Ca2+ oscillations continue for at least 60 min during Many cells that are operatedby calcium-mobilizing recep- sustained agonist stimulation(Stojilkovic et al., 1992b). Thus, release of sequestered Ca2+from intracellular pool(s) in the tors exhibit oscillatory [Ca2+Ii responses to agonist stimulation. The molecular mechanism(s) of such intracellular Ca2+ endoplasmic reticulum (ER) is the primary source of Ca2+ during the initial phase of the oscillatory response, whereas Ca2+ influx * The costs of publication of this article were defrayed in part by the steady-stateCa2+ oscillationsare dependent on the payment of page charges. This article must therefore be hereby during continued exposure to GnRH. marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $TOwhom correspondence and reprints request should be addressed ERRB, NICHD, Bldg. 49, Rm. 6A-36, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-2136;Fax: 301-480-8010.

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The abbreviations used are: Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; InsP3, inositol trisphosphate; GnRH, gonadotropin-releasing hormone; IP3R channels, inositol 1,4,5-trisphosphate receptor cbannels; ER, endoplasmic reticulum; TG, thapsigargin.

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[Ca2'Ii Oscillations in Gonadotrophs

Relatively little is known about the mechanism(s) of these agonist-induced Ca2+oscillations in pituitary gonadotrophs. Activation of protein kinase C by phorbol esters reduces the amplitude, but not thefrequency, of the agonist-induced Ca2+ transients. This suggests that an inhibi~ry action of protein kinase C on signal generation could occur during agonist stimulation but not as an integral part of the mechanism responsible for periodic release of intracellular Ca2+ (Stojilkovic et al., 1991). In cells exposed to high [K+],, the resulting depolarization-induced Ca2+influx elevates [Ca2+Iito a wide range of peak values, each followed byan exponential decrease to the steady-state level. However,such K+-inducedelevations of [Ca2+Iiare never associated with the development of an oscillatory response in pituitary gonadotrophs (Stojilkovic et al., 1991; Iida et ul., 1991). This finding argues against second messenger-controlled Ca2+oscillations in which the existence of two releasable Ca2+pools has been proposed, with calciuminduced calcium release as themechanism of transfer between the two pools (Goldbeter et al., 1990). We have further analyzed the mechanism(s) of Ca2+oscillations in agonist-stimulated gonadotrophs under several experimental paradigms. Our findings areconsistent with a second messenger controlled model of Ca2+oscillations, since the transients were observed in the presence of a relatively constant Ins(1,4,5)P3 concentration. In these cells, the degree of receptor occupancy, expressed as the Ins(1,4,5)P3concentration, determines the frequency of spiking. However, the inability of a GnRH antagonist and a phospholipase C inhibitor to alter the established pattern of GnRH-induced Ca2+ spiking reveals a differential requi~mentfor Ins( 1,4,5)P3 during the initiation and the maintenance of the oscillatory Ca2+response. EXPERIMENTAL PROCEDURES

The cells were continuously stimulated with the agents added in 1ml volumes dissolved in medium 199with 1.25 mM Ca2+,except those shown in Figs. 7 and lOE, which were stimulated underCa2+-deficient conditions ([Ca2+L200 nu). Thedoses shown in the figures are the f i a t dilutions of the drugs. There was sometimes a significant linear decrease in the fluorescence signal at both wavelengths during the recording period, but this did not exceed 5% in the majority of cells. Corrections for this decrease were not applied because the patternof Ca2+response based on ratio recording were identical in cells with negligible decrease of fluorescence and those with a substantial decrease. In accord with this, whole cell current recordings in patchclamped cells showed that the patterns of GnRH-induced Ca2+responses were similar in the presence and absence of Indo-1. V,,, and Whole Cell Current Recordings-Culture medium was replaced by a solution containing 145 mM NaC1, 4 mM KCI, 2.6 mM CaCl2,1mM M&l, 10 mM HEPES (pH adjusted to 7.35 with NaOH), 10 mM glucose. Cells were placed on the stage of an inverted microscope, and whole cell current was measured using the patch-clamp technique. Electrodes, pulled from soft capillary glass to obtain resistances 3 to 6 megaohms when measured in symmetrical 150 mM KC1 solution, were filled with a solution containing: 120 mM potassium aspartate, 20 mM KCI, 2 mM MgCIz,20 mM HEPES, pHadjusted to 7.2 with NaOH. Recordings were made using an EPC-7 patchclamp amplifier (List). Liquid junction potentials were compensated before touching the cell surface. Inositol phosphates were added from concentrated stocks made in the same solution. In some experiments, 2 mM adenosine trisphosphate (ATP) was added to the intracellular solution; we did not observe appreciable differences in the pattern, sensitivity to agonists, or time course of the oscillatory current responses among cells from which current was recorded using solutions with or without ATP. Once a gigaohm seal was obtained, the membrane patch under the electrode was broken by applying gentle suction. The currentsignal, recorded under voltage clamp, was filtered at 1kHz (low pass) using an 8-pole Bessel filter (Frequency Devices). Permanent records of membrane current were made on analog magnetic tape (Racal Recorders). All experiments were performed at 2024 "C. Calculations-The dependence of number of spikes on time, n ( t f , was evaluated by simple counting of spikes. A polynomial of third order, at3 + bt2 ct + d, was then fitted to n ( t ) by the method of least squares. Figs.9, B and C, show the first derivative of the polynomial with respect to time, 3at2 +2bt c, which represents the frequency of Ca2+spiking. The area under the [Ca2+]isignal uersus time course ( [Ca2+Ji dt) was calculated according to the trapezoidal rule. Integration limits are taken from the moment ( t l )prior to addition of stimulus to the time (t2)when [Ca2+Iireached the baseline value and showed no further spiking for about 60 s. The values shown in Fig. 7B are total surface area corrected by subtraction of the area under the base-line value.

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Chemicals-Indo 1-AM and Indo-1 free acid were purchased from Molecular Probes (Eugene, OR) and Calbiochem; thapsigargin was from L. C. Services (Boston, MA): U73122and U73433 wereprovided by Dr. E. Bleasdale, the Upjohn Company. GnRH and endothelin were from Peninsula Laboratories, Inc. (Belmont, CA).All other chemicals were obtained from Sigma. CytoplasmicCalcium Measurement-For [Caz+Jimeasurements, pituitary gonadotrophs from 2-week ovariectomized female rats (0.25 X 106/dish) were plated in 35-mm Petri dishes (Falcon) on 25-mm diameter glass cover slips (Erie Scientific Co.) coated with 0.01% poly-L-lysine (Sigma) and loaded with 2 PM Indo-1 AM. After incuRESULTS bation for 60 min at 37 "C, the coverslips were washed twice with fresh incubation medium and maintained in thesame medium in the ~ n t r a c e l l ~ ~ r f n ~ ( 1Concentration , 4 , 5 ) P ~ Determines the Patdark at 22 "C before fluorescence measurements. After washing, in- tern of the Ca2+Response-The dependence of the frequencydividual coverslips were transferred to a Leiden coverslip dish (Medical Systems Co., Greenvale, NY) with 1ml ofphenol red-free medium modulated Caz+response on amplitude-modulated changes in 199,mounted on the stage of an inverted Diaphot microscope attached Ins(1,4,5)P3levels was examined as follows. The cell interior to a dual emission microscopic fluorometer (Nikon, Garden City, wasexposed to Ins(1,4,5)P3, present in the pipette during NY), and examined at 24 "C under a X 40 or X 100 oil immersion patch-clamping, andthe oscillatory current response was fluorescence objective. For excitation of Indo-1, the light beam from recorded. GnRH has been shown to induce current oscillations a 100-watt mercury arc lamp was reduced by a 1/16th neutral density (Tse and Hille, 1992; Stojilkovic et al., 1992b) that are synfilter and passed through a 360-nm interference filter andthen reflected from a DM 400 dichroic mirror through a pinhole diaphragm chronized with and dependent on [Ca'+]i oscillations (Kukulthat was slightly smaller than the selected cell. The emergent beam jan et ai., 1992). Such synchronization of the membrane was split by a DM 455 dichroic mirror and directed through 405- and current with Ca2+ transients in gonadotrophs is similar to 485-nm interference filters with a 20-nm band pass. Each beam was that observed in other cells operated by calcium-mobili~ng monitored by an individual photomultiplier tube, and the ratio of receptors (Osipchuk et al., 1990; Parker andIvorra, 1990) and intensities was calculated by a computer at 360-ms intervals through- provides an indicator of the Ca2+ response to agonists and out the experiments, using the FASTINCA program (University of other stimuli. Intracellular application of Ins(1,4,5)P3has also Cincinnati Medical Center, Cincinnati, OH). AI1 [Caz+Jivalues were derived from a standard curve that was been shown to mimic agonist-induced current oscillations constructed by addition of increasing concentrations of Ca2+ (10- (Tseand Hille, 1992; Wakui et aL, 1989), consistent with 1500 nM) to 15 p~ Indo-1 free acid. For background collection, the second messenger-controlled Ca'+ oscillation models that do dual emission of an open area of the culture dish with medium 199- not require fluctuations in Ins(1,4,5)P3. Hanks' was recorded, since the intensity of the resulting fluorescence In gonadotrophs, three types of current responses (Fig. 1, was almost identical to that of unloaded cells. After subtraction of the background from the emission of the specimen, the ratios of the right panels) were observed in cellsexposed to increasing net values were applied to the standard curve stored in the computer. Ins(1,4,5)P3concentrations: subthreshold (1st panel);thresh-

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[Ca2+]i Oscillations in Gonadotroph 1os

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current

lOPA

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100 pM

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Ins (1,4,5)P3,200 pM FIG. 2. Membrane current responses to Ins(1,4,5)Psand a nonmetabolizable analog. The data shown are representative of

FIG. 1. Comparison of [Cas'], and membrane current oscillations in gonadotrophs stimulated by gonadotropin-releasing hormone ( G n R H ) and Ins(1,4,5)PS.Left panels, typical [Ca2+li four experiments for each group. profiles from gonadotrophs exposed to increasing concentrations of GnRH. In this and the following figures, the arrows indicate the times of agonist application. Cells were continuously exposed to agonists from the moment of application to the end of the experiments. The data shown are representative of 300 records from single gonadotrophs. Right panels, whole cell membrane current oscillations induced by Ins(1,4,5)P3.The concentrations of Ins(1,4,5)P3 indicated below the tracing are intrapipette concentrations. The data shown are representative of four to six experiments for each dose. The axes are identical for all records.

was not altered (Fig. 3A). Similarly, a progressive increase in antagonist concentration during GnRH-induced Ca2+spiking did not alter the frequency and amplitude of the Ca2+transients (Fig. 3, D versus E ) . As expected, low concentrations of GnRH (0.1 (not shown) and 1 nM (Fig. 4C)) did not initiate oscillatory Ca2+responses in cells previously exposed to 1 W M antagonist. Further increases in agonist concentration elicited frequency-modulated old-oscillatory, with modulation of the frequency of current Ca2+signaling, but of much lower rate than in controls (Fig. oscillations to increasing Ins(1,4,5)P3concentrations andpro- 4, A versus C). A decrease in GnRH antagonist concentration to 100 nM was followed by a proportional shift in sensitivity gressive decrease in their amplitudes (2nd to 4th panels);and to subsequent stimulation by GnRH (Fig. 4B). threshold-biphasic, composed of the spike phase followed by The potent phospholipase C inhibitor, U73122 (Bleasdale a sustained oscillatory plateau phase (5th panel).Such patet al., 1990;Yule and Williams, 1992), was also utilized to terns of current responses are indistinguishable from the Ca2+ determine the dependence of Ca2+ spiking on initialand responses of intact gonadotrophs stimulated with increasing sustained changes in phosphinositide hydrolysis. In cells exGnRHconcentrations (leftpanels). The nonmetabolizable posed to the inhibitor for 100 s, the Ca2+response to subseIns( 1,4,5)P3 analog, 3-deoxy-3 fluoro-D-myo-inositol 1,4,5- quent stimulation by 0.5 nM GnRH was reduced in a dosetrisphosphate(F-IP3), induced similar patterns of current dependent manner, with an ICso of 4 WM, and was abolished oscillations (Fig. 2), in agreement with the hypothesis that a at 10 W M U73122 (Fig. 5 , A and B ) . In control cells, the relatively constant Ins( 1,4,5)P3 level initiates the oscillatory GnRH-induced Caz+spiking response was unchanged by exCa2+release mechanism. Thus, second messenger-controlled posure to the inactive compound, U73443 (Fig. 5C). In conmodels are clearly applicable to the analysis of agonist-in- trast to the prominent inhibitory action of U73122 on the duced Caz+oscillations in gonadotrophs. GnRH-induced Ca2+signal, the frequency and amplitude of Differential Requirements for Ins(1,4,5)P3 during Initiation established Ca2+ transients in GnRH-stimulated cells were and Maintenance of Ca2+Spiking-The roles of the initial and not altered by subsequent application of 10 PM U73122 (Fig. sustained levels of GnRH receptor occupancy (and consequent 6). Ins(1,4,5)P3 formation)in the pattern of Ca2+signaling were These data argue against receptor-controlled models of Ca2+ examined in gonadotrophs in which transientstimulation transients, which require that oscillations of [Ins(1,4,5)P3Ii with GnRH was terminated by addition of the potent GnRH and [Ca2+]ibe in phase, and support second messenger-con[~-acety~-~-p-chloro-Phe'~~,~-Trp~,~-Lys~,~antagonist trolled models in which the frequency of Caz+ spiking is Ala'OIGnRH. This compound has been shown to cause a determined by the level of receptor occupancy, i.e. by the prompt(within15 s) decrease in Ins(1,4,5)P3 production resulting concentration of nonoscillating Ins(1,4,5)P3. Howtoward the basal level when applied in 100-fold excess after ever, the pattern of Ca2+spiking is no longer dependent on GnRH (Morgan etal., 1987). However, itsadministration the level of Ins(1,4,5)P3when the process of repetitive intraafter GnRHdid not change the patternof the agonist-induced cellular Ca2+release has begun. This suggests that the initial Ca2+response (Fig. 3C). The frequency and amplitude of Ca2+ interaction between Ins(1,4,5)P3 and itsreceptors determines spiking were also unaffected by addition of a 1,000-foldexcess the frequency of transientsina concentration-dependent of the antagonist,although the duration of signaling was manner; this frequency can be increased, but not decreased, shortened (Fig. 3B). Addition of a 10,000-fold excess of the by subsequent changes in Ins(1,4,5)P3production. Such obantagonist also caused a relatively prompt decrease in the servations are in accord with second messenger-controlled duration of Ca2+signaling, but again the patternof oscillations models of oscillatory Ca2+signaling based on a self-sustained

[Ca2+/iOscillations in Gonadotroph

7716

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100 200 Time (seconds)

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FIG. 4. Modulation of Ca2+spiking frequency by increases in receptor occupancy. A , agonist-induced Ca2+transients in controls. B and C, increases in frequency of Ca2+spiking in cells previously exposed to the GnRH antagonist, followedby application of increasing GnRH concentrations.

FIG. 3. Maintenance of agonist-induced Ca2+ spiking frequency and amplitude in antagonist-treated gonadotrophs. A potent GnRH antagonist [~-acetyl-~-p-chloro-Phe'~~,D-Trp~,~proportional to the extent of Ca2+release from the agonistL ~ S ' , D - A ~ ~ ' ~ ] G(GnRH-An.) ~RH was added after the initiation of sensitive Ca2+pool. The measurement of intraspike area thus [Ca2+Iiresponses by GnRH. The initial GnRH concentrations were diluted after addition of GnRH-An to two-thirds of the initial level. provides an index of the efficacy with which specific GnRH concentrations release sequestered Ca2+ from the agonistThe data shown are representative of 20 independent experiments. sensitive pool. The intraspike area of the Ca2+response progressively increased over a low agonist dose range, reaching a autocatalytic process that is triggered by Ins(1,4,5)P3. maximum at 0.1 nM GnRH; further increases in GnRH conEffects of Cytoplasmic and Luminal Ca2+on Agonist-induced centration were ineffective (Fig. 7 B ) .It is interesting that the Ca2+Spiking-At the threshold agonist dose, the frequency threshold dose for GnRH (defined as the minimum concenof Ca2+spiking was low and irregular (Fig. 1, GnRH: 10 p ~ ) . tration required for initiation of Ca2+ spiking) (Iida et al., The amplitudes of such transients were also variable, and no 1991) released about 50%of the sequestered Ca2+,as indicated strict relationship between the frequency and amplitude of by the arrow in Fig. 7B. transients could be observed. However, at higher agonist doses The residual sequestered Ca2+was estimated by addition of the frequency and amplitude of the Ca2+transients were inter- 5 PM ionomycin, a dose that mimics agonist-induced depletion related, such that increases in frequency due to increases in of the intracellular Ca2+ pool(s) (Stojilkovic et al., 1992b). agonist concentration were accompanied by a more rapid Application of the ionophore 7 min after a wide range of decrease in spiking amplitude (Fig. 7A).To determine GnRH doses (0.1-100nM) caused little additional Ca2+rewhether the depletion of the agonist-sensitive Ca2+pool or lease, equivalent to less than 5% of the amplitude and less attenuation of the spread of the Ca2+signal within the cell is than 3% of the total intraspike area of the Ca2+responses in responsible for this decline in the amplitude of Ca2+spiking, cells exposed to theionophore alone (not shown). These data we performed two additional experiments. suggest that the total release of the sequestered Ca2+occurs First, gonadotrophs were exposed continuously (for 7 min) over a wide range of agonist doses and that higher agonist to increasing doses of GnRH and the total intraspike area concentrations induce more rapid depletion of the agonistwas calculated. In the absence of Ca2+ entry, such area is sensitive Ca2+pool.

[Ca2+]i Oscillations in Gonadotrophs

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U73343.10pM GnRH. 0.5nY

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FIG. 5. Dose-dependenteffect of the phospholipaseC inhibitor, U73122, on the frequency of GnRH-induced Ca2+ spiking. A, the tracings shownare typical for U73122-induced modulation of Ca2+signaling in GnRH (0.5 nM)-stimulatedgonadotrophs. B , averaged data from three tofive measurements per dose. C, inability of the inactive isomer, U73343, to abolish GnRH (0.5 nM)-induced Ca2+spiking.

=.

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t

t

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GnRH U73122 0.5M 10pM

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FIG. 6. Maintenance of agonist-induced Caa+ spiking frequency and amplitude in U73122-treated gonadotrophs. A, controls; B , U73122-treatedcells.Thetracings are typical of 10 experiments with similar results.

Second, we determined whether the decreasing Ca2+content of the agonist-sensitive pool affects the amplitude of spiking similarly in different regions of the cell during GnRH stimulation. As shown in Fig. 8B,the pattern of the Ca2+response was unaffected when the area of the cell from which the Ca2+ response was recorded was reduced from 100 to 25%, and subsequently to 6%. Whenthe Ca2+signal was recorded from only 6% of the totalcell surface, the pattern of Ca2+response was independent of the location of the recording area (center, close to plasma membrane, or in between; not shown). Thus, these data suggest that the decrease in the amplitude of Ca2+ spiking is not related to the inability of the Ca2+waves to spread within the cells, further confirming that progressive depletion of the agonist-sensitive Ca2+ pool underlies the decrease in the spiking amplitude.

7717

In the presence of a constant GnRH concentration, the decrease in theamplitude of Ca2+spiking was associated with a progressive increase in spiking frequency. As shown in Fig. 9A, the time required for 10 spikes decreased progressively during the initial predominantly extracellular Ca2+-independent phase. During the sustained extracellular Ca2+-dependent phase, the frequency of spiking remained relatively high. For more detailed analysis of this trend, the frequency of Ca2+ spiking was derived utilizing the cumulative number of spikes per time in cells continuously exposed to GnRH. The data illustrated in Fig. 9B show that the frequency of Ca2+transients progressively increased during continuous stimulation Yith GnRH (0.1 nM), not only in the presence, but also in the absence, of extracellular Ca2+. Furthermore, application of the qhospholipase C inhibitor, U73122, did not affect the increase in Ca2+ spiking frequency in cells stimulated with 0.5 nM GnRH (Fig. 9C). Since the increase in spiking frequency is not related the the sustained Ins(1,4,5)P3concentration, but temporally correlates with a decrease in luminal Ca2+, it is reasonable to postulate that intra-ER calcium inhibits IP3R channels and that initiation of Ca2+release overcomes such inhibition. In accord with this view, the endoplasmic reticulum (Ca'+)ATPase blocker, thapsigargin (TG), induced oscillatory Ca2+ response in pituitary gonadotrophs. TG prevents compensation of the spontaneous leakage of Ca2+ from the agonistsensitive Ca2+pool(s) due to its concentration gradient between the ER and cytoplasm. In gonadotrophs, thapsigargin elicited three types of [Ca2+Iiresponses: subthreshold, oscillatory, or nonoscillatory biphasic (Fig. 10, A-C) in the presence (D)or absence ( E ) of extracellular Ca2+.In addition, such TG-induced Caz+ release markedly attenuated the response to subsequent agonist stimulation ( F ) . However, the Ca2+ transients induced by TG, unlike the base-line Ca2+ spiking induced by GnRH, took the form of low-frequency sinusoidal-like oscillations. The inability of TG to induce typical base-line Ca2+ spiking is not unexpected, since the drug attenuatesthe oscillatory Ca2+ response in agoniststimulated cells (Iida et al., 1991). The types of Ca2+responses elicited by TG were markedly dose-dependent. In the low dose range (1-50 nM), TG caused subthreshold Ca2+responses (12 of 20) and only occasionally a threshold-oscillatory response (2 of 20). The rest of cells did not show obviouschanges in the basal [Ca2+Ii.A t higher doses (0.1-1 HM), an increase in [Ca2+Iiwas observed in all stimulated cells; the drug induced oscillatory responses in 11 (20%) cells, biphasic responses in 2 (4%)of 55 cellsand subthreshold , responses in the rest. At high doses (5-10 p ~ ) oscillatory responses were observed in 34 (65%) cells and biphasic responses in 6 (11%)of52 cells. Incontrast, caffeine was practically ineffective in initiating the oscillatory Ca2+ response; only 2 of 115 (2%) stimulated cellsshowed Ca2+ spiking. Also, highK+-induceddepolarization and stimulation with calcium channel agonist Bay K 8644 were never associated with initiation of oscillatory Ca2+responses (not shown), although all stimulated cells (150 cells with KC1 and 14 cells with Bay K 8644) showed concentration-dependent rises in [Ca2+Iiin the range comparable with GnRH-stimulated cells. These observations argue against the idea that Ca2+oscillationsare mediated by the Ca2+-dependent activation of phospholipase C (reviewed in Davis, 1992), since rises in [Ca2+Iishould lead to the activation of phospholipase C independent of the source of ion. The finding that Bay K 8644induced Ca2+ signaling spreads throughout gonadotrophs (Rawlings et al., 1991) negates the possibility that regional Ca2+entry through voltage-sensitive calcium channels does

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[Ca2+JiOscillations in Gonadotrophs

B 50-

FIG.7. Frequency and amplitude of Ca2+ spiking in agonist-stimulated gonadotrophs.A , typical profiles

40-

of Ca2+responses in cells continuously exposed to GnRH (0.05, 0.1, 1, and 10 nM, from top to bottom) for 7 min, under extracellular Ca2+-deficient conditions [Ca2+], = 200nM. B, relationship between agonist dose and the total intraspike area. The values are the means S.E.of the numbers of observations indicated above the data points. The intraspike areawas calculated as described under "Experimental Procedures." The arrows indicate the threshold dose for GnRH, defined as the minimum dose required to trigger the oscillatory response.

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FIG.8. Independence of the amplitude of transients from the total cell area from which [Ca2+]iwas recorded. A , 410-and 485-nm output during the recording. A pinhole diaphragm was initially adjusted to thesize of the selected cell and was later reduced to 25 and 5 % of the totalcell area. B , corresponding Caz+concentrations calculated from the ratio of intensities at 360-ms intervals, applied to the standard curve. The recording was obtained with a X 100 objective.

not have access to phospholipase C. Furthermore, it is most unlikely that TG-induced activation of oscillatory Ca2+responses is related to a rise in Ins(1,4,5)P3,since TG does not stimulate inositol phosphate production in several cell types, although in some cells it can initiate oscillatory responses (Jackson et al., 1988; Takemura et al., 1989; Kwanet al., 1990; Law et al., 1990; Ely et al., 1991; Foskett et al., 1991). On the other hand, these data satisfy a model based on the negative

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FIG.9. Increase in the frequency of Cas+ spiking during agonist stimulation. A , the gradual decrease in the spike interval per 10 spikes indicates the increase in the frequency of Ca2+signaling. B , frequency of GnRH (0.1 nM)-induced Caz+spiking in the presence of 1.25 mM Ca2+and in Ca2+-deficientmedium ([Ca"], = 200 nM). c, frequency ofCa" oscillations in response to 0.5 nM GnRH in the absence and presence of 10 PM U73122 (arrow indicates moment of application of the drug).

effects of luminal Ca2' on IP3R channel opening, as well as an autocatalytic mechanism of Ca2+ spiking in which Ins(1,4,5)P3is not an essential requirement. DISCUSSION

These observations in pituitary gonadotrophs are consistent with a mechanism of Ca2+ oscillations in which

[Ca"]i Oscillations in Gonadotrophs

7719

Ins(1,4,5)P3level in a self-sustained autocatalytic manner. (i) The established Ca2+ spiking frequency is independent of the magnitude of the sustainedIns(1,4,5)P3 response (up- but not down-regulation of the spiking frequency). (ii) Thedegree of receptor occupancy, i.e. the prevailing Ins(1,4,5)P3 concentration, determines the rate, but not the extent,of Ca2+release; once initiated, Ca2+release continues until the agonist-sensitive Caz+pool is depleted. (iii) As in parotid cells (Foskett et al., 1991), thapsigargin can induce oscillatory Ca2+responses of similar characteristics to thosetriggered by agonist stimulation. These observations indicate that the status of IP3R channels is not exclusively regulated by Ins(1,4,5)P3. Since promoters of Ca2+ release or leakage from the ER can initiate oscillatory responses, and elevation of [Ca"], per se cannot, it is reasonable to speculate that the Ins(l,4,5)P3-activated ULJ J Ca2+ channelsareunderintra-ERinhibition, presumably t t t mediated by the high luminal Ca'+ concentrations. AgonistTG. 5pM TG. 1pM CnRH.100nM stimulated production of Ins(1,4,5)P3would oppose such inhibition by promotingCa2+ discharge,presumablyin that 0 100 200 300 0 100 200 300 region of the ER adjacent to the plasma membrane, where Seconds FIG. 10. Effects of the (Ca2+)-ATPaseblocker, thapsigargin the concentrations of Ins(1,4,5)P3 arehighest. As a result, ER (Tc),on [Caa+liresponses. Leftpanel, subthreshold ( A ) ,oscillatory [Ca'+] would decrease in this and adjacent areas and thus ( B ) ,and nonoscillatory biphasic(C) responses in gonadotrophsstim- further facilitate Ca2+ release. In the case of threshold Ca2+ ulatedwithTG. Right panel, TG-induced Ca2+ transients in the release, the decrease in luminal [Ca"] renders the residual presence of 1.25 mM Ca2+ (D)and in Ca2+-deficientmedium ( E ) . IP3Rchannels along the ER operative in the absence of Depletion of the agonist-sensitiveCa2+pool during TG-inducedele- Ins(1,4,5)P3,leading to progressive recruitment of calcium vation of [Ca2+IL, indicated by attenuation of the GnRH-induced release. Since low concentrations of thapsigargin also selec[Ca2+];responses ( F ) . tively deplete the [Ca2+IER, itlikewise can activate an oscillatory response. Ins(1,4,5)P3 levels donotfluctuate, sincecellsexposed to Such negative control of Ca2+discharge through IP3R chanrelatively constant levels of Ins(1,4,5)P3or its active analogs nels is a major factor in one of the computer simulations of show prominent oscillatory responses. The patterns of Ca*+ Ca2+oscillations (Swillens and Mercan, 1990). On the other response induced by Ins( 1,4,5)P3 and agonist stimulation are hand, it has been suggested that the decreasing luminal Ca2+ indistinguishable, each including the subthreshold response, contentretardsfurtherIns(l,4,5)P3-inducedCa2+ release the threshold-oscillatory response with modulation of spiking (Missiaen et al., 1992),suggestingcooperativitybetween frequency by increasing Ins(1,4,5)P3 concentrations, and the Ins(1,4,5)P3 and luminal Ca2+ on opening the of IP3R channels summation of individual spikes intoa biphasic response.Also, (Nunn and Taylor, 1992; Missiaen et al., 1992). These data theamplitude of Ca2+spikingduringIns(l,4,5)P3-induced Ca2+responses is inversely related to thefrequency of spiking also suggest a continuous steady-state mechanism of Ca'+ release, in contrast to observations onpurified IP3R channel in a manner similar to that observed in agonist-stimulated gonadotrophs. Thesefindingsindicatethatthepattern of molecules reconstituted into lipid vesicles, which respond in intracellular Ca2+ signaling in gonadotrophs is determined by a quantal manner (Ferris et ab, 1992). Our data do not support the concept of steady-state Ca2+ the magnitude of the Ins(1,4,5)P3 response to GnRH. The ( a ) In gonadotrophs, comrelease from two crucial aspects. ability of anonmetabolizable InsP3analogto elicit Ca2+ plete Ca2+ discharge occurs in a wide range of agonist concentransients further indicates that oscillations in Ins( 1,4,5)P3 trations, whereas only partial release was observed from perare not required to induce Caz+ spiking in gonadotrophs. In addition, the present data provide several lines of evi- meabilized A7r5 cells, in which the decrease in the luminal dence that the kinetics of endogenous Ins(1,4,5)P3 production level of Ca2+ limits furtherion release (Missiaen et al., 1992). and of Ca2+discharge during prolonged agonist stimulation ( b ) The consequence of a steady-state model is thatthe are not closely correlated rather, intracellular Ca2+signaling frequency of transients should decrease with continuation of is composed of two distinct phases,initiation and continuation Ca'+ spiking due to the progressive decrease in [Ca2+IER. In of Ca2+spiking, with differential sensitivity to the magnitude contrast to this, the present data demonstrate that the freof the Ins( 1,4,5)P3 response. Under physiological conditions, quency of spiking progressively increases during sustained the initiation of Ca2+ spiking is dependent on the increase in intracellular Ca2+ signaling, indicating that decrease in luminal [Ca"] furtherfacilitatesCa2+ release. The negative Ins(1,4,5)P3 production that occurs during agonist stimulation. In gonadotrophs, the competitive Ins( 1,4,5)P3-receptor control of Ca2+release by luminal Ca2+may serve as a stabiantagonist, heparin, prevents the initiationof the oscillatory lizing factor in unstimulated cells, by protecting them from Ins(1,4,5)P3;this response by GnRH, and thisblock can beovercome by higher activation by backgroundoscillationsin agonist concentrations (Tse and Hille, 1992). Dynamic video- would be consistent with the low frequency of TG-induced gonadotrophs. However, once a threshold imaging Ca2+ microscopy has shown that initiation of the oscillationsin GnRH-induced [Ca"], response occurs at a specific locus of amount of Ca2+ is released, such a system would further the gonadotroph,close to the plasma membrane(Rawlings et facilitate Ca2+ release, even when the Ins(1,4,5)P3 level deal., 1991). This could correspondtothesubthresholdrecreases, rendering the system less dependentonsustained sponses observed in single-cell fluorometric studies (Iida et Ins(1,4,5)P3 elevations. For example, GnRH induces a bial., 1991). phasic Ins(1,4,5)P3response (Morgan et d . , 1987) and endoOnce initiated, however, Ca2+spiking canoccur at the basal thelin evokes only a monophasic response in pituitary cells, L

J

7720

[Ca"]i Oscillations in Gonadotrophs open state. On the other hand, a decrease in receptor occupancy during agonist stimulation does not affect the frequency of oscillations, since the initial level of depletion of [Ca'+],, determines the statusof IP3R channels. The luminal inhibition of IP3R channel activity is sufficient to explain the receptor- and nonreceptor-mediated initiation of Ca2+ spiking and modulation of frequency of Ca2+ signaling, but not the autocatalytic oscillatory nature of the Ca2+ response per se. In our model, the essential factors for Ca2+ spiking, in addition to the decrease in the [Ca2'li-mediated inhibition of IP3R channel conductance that renders them independent of IP3, are alternating refractoriness andregeneration of signaling mediated by [Ca2+Ii and (Ca2+)-ATPase. As shown in Fig. 11C, the rise in [Ca2+Iiinhibits further Ca2+ release due to a stilluncharacterized mechanism,whereas (Ca2+)-ATPaseopposes such inhibition by promoting reuptake of Ca2+by the ER (Fig. l l D ) . I

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FIG. 11. Model of Ca2+oscillations. In this "one-calcium pool" model, oscillatory Ca2+ responses are generated at aconstant Ins(1,4,5)P3 level by bidirectional (intra-ER and cytoplasmic) inhibition of IP3R channels by Caz+ and by the action of ER (Ca")ATPase. Central panel, diagram of the interacting elements involved in the oscillatory Ca2+response during the initiation ( l e f t ) and propagation (right) of signaling. Dottedarrows, negative effects; full arrows, positive effects. Cornerpanek, hypothetical curves describing the individual interactions. A , in unstimulated cells, IP3R channels are under constant Ca2+-mediatedinhibition from the luminal aspect of the ER membrane. B, InsPB initiates signaling by triggering the flow of Ca2+from ER to cytoplasm. The resulting decrease in luminal [Ca"] renders the residual IP3R channels along the ER operative in the absence of Ins(1,4,5)P3. C , the rise in [Ca"], bidirectionally modulates IP3R channelconductance and stimulates (Ca2+)-ATPase. D,(Ca2+)-ATPaselowers [Ca2+Iibelow the critical inhibitory level and the channels again conduct Ca2+into the cytoplasm.

due to rapid desensitization (Stojilkovic et al., 1992a), but both agonists induce similar patterns of oscillatory Ca2+ response (Stojilkovic et al., 1990). At present, however, it is not clear whether such release requires a basal level of Ins(1,4,5)P3, or if theIP3Rchannelsremainopeninthe absence of Ins(1,4,5)P3 during the sustainedresponse due to the decrease in luminal Ca2+. Based ontheseandotherobservations, we propose an integrated model of Ca2+ spiking in gonadotrophs. The elements involved in initiation and propagation of Ca2+signaling are illustrated in the central panel in Fig. 11. In this model the statusof the IP3R channels is determined by Ins(1,4,5)P3 and luminal Ca2+ ( A and B ) . Thus, the frequency of Ca2+ spiking is initially dependent on Ins( 1,4,5)P3 concentration and subsequently on [Ca2+IER.An increase in receptor occupancy, i.e. production of Ins(1,4,5)P3, increases thefrequency of Ca2+ spikingby increasing the number of channels in the

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