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Am J Physiol Cell Physiol 282: C635-C645, 2002. First published November 7, 2001; doi:10.1152/ajpcell.00044.2001
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Vol. 282, Issue 3, C635-C645, March 2002

Caffeine-stimulated GTH-II release involves Ca2+ stores with novel properties

James D. Johnson*, Calvin J. H. Wong*, Warren K. Yunker, and John P. Chang

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Modulation of Ca2+ stores with 10 mM caffeine stimulates robust secretion of gonadotropin (GTH-II) from goldfish gonadotropes. Although both endogenous forms of gonadotropin-releasing hormone (GnRH) utilize a common intracellular Ca2+ store, sGnRH, but not cGnRH-II, uses an additional caffeine-sensitive mechanism. We examined caffeine signaling by using Ca2+ imaging, electrophysiology, and cell-column perifusion. Although caffeine inhibited K+ channels, this action appeared to be unrelated to caffeine-induced GTH-II release, because the latter was insensitive to tetraethylammonium. The effects of caffeine also were not mediated by the cAMP/protein kinase A pathway. Instead, caffeine-evoked GTH-II responses were Ca2+ signal dependent because they were abolished by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid loading. Caffeine generated localized Ca2+ signals that began near secretory granules. Surprisingly, caffeine-stimulated GTH-II release was insensitive to 100 µM ryanodine and, unlike GnRH action, was unaffected by inhibitors of voltage-gated Ca2+ channels or sarco(endo)plasmic reticulum Ca2+-ATPases. Collectively, these data indicate that caffeine-stimulated GTH-II release is not mediated by typical agonist-sensitive Ca2+ stores found in endoplasmic reticulum.

ryanodine; sarco(endo)plasmic reticulum calcium adenosine triphosphatase; potassium channels; voltage-gated calcium channels; protein kinase A; secretory granules


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INTRACELLULAR CA2+ stores are important components of the signal transduction of many neuroendocrine regulators. Although multiple Ca2+ stores have been colocalized to the endoplasmic reticulum (ER), several other organelles, such as Golgi and secretory granules, contain considerable quantities of Ca2+ (41) and may act as atypical agonist-sensitive Ca2+ pools in secretory cells (13, 21, 25, 32, 36, 37, 39). Multiple independent intracellular Ca2+ stores have been identified in a variety of cell types (15, 18). Intracellular Ca2+ stores including, but not limited to, those sensitive to inositol 1,4,5-trisphosphate (IP3), ryanodine (cADPribose sensitive), and nicotinic acid adenine dinucleotide phosphate have been characterized biochemically (Refs. 3, 4, 12, 30; reviewed in Ref. 21). One of the most valuable agents for elucidating the roles of intracellular Ca2+ stores in physiological processes has been caffeine because it is membrane permeant and activates ryanodine-sensitive Ca2+ release channels by sensitizing them to ambient Ca2+ levels (34, 41).

We have shown that 10 mM caffeine evokes robust secretion of maturational gonadotropin hormone (GTH-II) from cultured goldfish gonadotropes (23). Particularly, caffeine treatment abolished the GTH-II-releasing effect of one endogenous form of gonadotropin-releasing hormone (sGnRH) but not the other (cGnRH-II). Although the acute signaling cascades of both neuropeptides are entirely dependent on intracellular Ca2+ stores (23), other studies have revealed that sGnRH, but not cGnRH-II, mobilizes depletion-resistant intracellular Ca2+ stores and generates IP3 (7, 19). The physiological relevance of these differences in signal transduction is underscored by the demonstration that these two closely related endogenous neuropeptides can generate quantitatively distinct Ca2+ signals in identified gonadotropes (24) and differentially regulate GTH-II gene expression (27). Interestingly, over the course of the yearly reproductive cycle, the efficacy of caffeine-stimulated GTH-II release parallels that of sGnRH (20). Can caffeine be used as a probe for the selective study of sGnRH Ca2+ signaling in goldfish gonadotropes? Before this question can be answered, a deeper understanding of the mechanisms of caffeine-stimulated GTH-II release must be ascertained. Thus we have examined the features of caffeine-dependent signal transduction in goldfish gonadotropes by using single-cell Ca2+ imaging, patch-clamp electrophysiology, and cell-column perifusion of cultured goldfish pituitary cells.

We have demonstrated that caffeine-evoked GTH-II release is resistant to blockade of extracellular Ca2+ entry through voltage-gated Ca2+ channels (VGCC) but is completely dependent on the ability of caffeine to generate Ca2+ signals. Together with the characteristics of caffeine-stimulated GTH-II release, the features of caffeine-evoked Ca2+ signals suggest the possibility that caffeine mobilizes Ca2+ from novel, localized stores in or around the secretory granules. Unlike the situation in other cells types, caffeine-stimulated hormone release was independent of the modulation of K+ current, ryanodine receptors (RyR), or the sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs). Thus caffeine may activate an intracellular Ca2+ store that is novel in both subcellular location and pharmacology in goldfish gonadotropes.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. High-purity ryanodine (99.5%), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), N-[2-(p-bromocynnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), and 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) from Calbiochem (La Jolla, CA) and dantrolene from RBI (Natick, MD) were dissolved in dimethyl sulfoxide (DMSO). Caffeine (RBI) was dissolved directly into media. At the concentration used in this study (0.1%), DMSO does not affect GTH-II release, intracellular Ca2+ concentration ([Ca2+]i), or ionic currents (not shown).

Animals and cell preparation. Animal use protocols were approved by the University of Alberta Biological Sciences Animal Care Committee in accordance with national guidelines. Goldfish from Aquatic Imports (Calgary, Canada) were maintained as described previously (5). Animals were anesthetized in 0.05% tricaine methanesulfonate before decapitation and removal of the pituitary. Cells were dispersed enzymatically and cultured overnight on Cytodex beads (for perifusion), 24-well surface-modified (Falcon Primaria line) tissue culture plates at a density of 0.25 million cells/well (for 2-h static incubation), or polylysine-coated coverslips at a density of 0.25 million cells/well (for Ca2+ imaging and electrophysiology) in medium 199 (M199) with Earle's salts (GIBCO, Grand Island, NY) supplemented with 2.2 g/l NaHCO3, 25 mM HEPES, 100,000 U/l penicillin, 100 mg/l streptomycin, and 1% horse serum, pH adjusted to 7.2 with NaOH, and cultured overnight at 28°C, 5% CO2, and saturated humidity. Additional details are published elsewhere (5, 24).

Cell identification. Gonadotropes were identified under Nomarski differential interference contrast (DIC) microscopy optics by using a validated set of morphological criteria, which previously was shown to allow the correct identification of GTH-II-containing and GnRH-sensitive cells with >95% accuracy (24, 50). The morphologically identifiable subpopulation of gonadotropes represents approximately half of all GTH-II-containing cells in the dispersed goldfish pituitary (24). Before imaging, the location of visible landmarks, such as the reniform nucleus located against one side of the cell and groups of large globules and secretory granules generally found opposite the nucleus, were carefully mapped out.

Fura 2 cytosolic Ca2+ imaging. Cells were evenly loaded with fura 2 by 35-min incubation in imaging medium [M199 with Hanks' salts (GIBCO), without phenol red, supplemented with 2.2 g/l NaHCO3, 25 mM HEPES, 100,000 U/l penicillin, 100 mg/l streptomycin, 0.1% BSA; pH adjusted to 7.2 with NaOH] at 28°C and 5% CO2 in 10 µM fura 2-AM (Molecular Probes, Eugene, OR), which had been prepared as a 1,000× stock solution in DMSO with 20% (vol/vol) Pluronic F-127 (24). After this incubation, cells were washed three times over a 15-min period. Cells were constantly perifused by using a pump-driven system at a rate of 1 ml/min with imaging medium at room temperature (~20°C). The bath volume was <100 µl, and the inflow was placed close to the cell of interest; complete solution changes were achieved in the vicinity of the cell in ~10 s.

Fura 2, in cells that had been selected with the morphological criteria, was excited with a Hg-Xe arc lamp (Hamamatsu) at 340- and 380-nm wavelengths (200 ms each) by using a computer-controlled filter wheel (Empix Imaging, Mississauga, ON). Emission fluorescence, at 510 nm, was recorded through the ×100 oil-immersion objective (1.3 NA Fluor) of a Zeiss inverted microscope (Carl Zeiss Canada, Don Mills, ON) with a Paultek Imaging intensified charge-coupled device video camera (Grass Valley, CA). Because of the long duration of the experiments, the image capture frequency did not exceed 10 s, to minimize photobleaching.

Ca2+ signals were analyzed off-line with a Macintosh G3 computer by using the public domain program NIH Image and Ca2+Ratiometrics 1.3, written by C. J. H. Wong. This macro automatically subtracts background fluorescence and thresholds the image. The emission intensity ratios (340-nm vs. 380-nm excitation) were converted to [Ca2+]i estimates by using the formula of Grynkiewicz et al. (16) and empirically derived constants as detailed previously (24).

Electrophysiology. Gonadotropes were morphologically identified as described in Cell identification. After 24-h incubation, cells were rinsed three times in testing medium and then perifused on the stage of an inverted microscope for 20 min before being recorded at room temperature (~20°C) as described previously for our laboratory (48). Fire-polished pipettes were filled with (in mM) 120 K+-aspartate, 20 KCl, 20 HEPES, 2 MgCl2, 1 CaCl2, 10 EGTA, 2.5 Na2ATP, and 0.4 Na2GTP, with pH adjusted to 7.2 with 1 M KOH. Bath solution was standard testing medium. Open-tip resistance was 3-5 MOmega . Series resistance (10-20 MOmega ) was compensated ~80%. After the whole cell configuration was obtained (17), the extracellular solution was switched over to testing medium containing TTX (100 nM) and Cd2+ (50 µM) to block Na+ and Ca2+ conductances, respectively. The liquid junctional potential (~10 mV) between the pipette and the bath solution was corrected for using post hoc methods (2). Currents were recorded with a Dagan 3900 patch-clamp amplifier, a Digidata 1200 acquisition board, and pCLAMP 5.2 (Axon Instruments, Foster City, CA). Leak and capacitive currents were subtracted using the P/4 procedure. Data were analyzed by using custom-written macros in Igor Pro (Wavemetrics, Lake Oswego, OR) or Excel (Microsoft, Seattle, WA).

Hormone release. For dynamic measurements of hormone secretion, cells grown on Cytodex beads (1.5 million/column) were perifused at 18°C with testing medium (M199 with Hanks' salts, supplemented with 2.2 g/l NaHCO3, 25 mM HEPES, 100,000 U/l penicillin, 100 mg/l streptomycin, 20 mg/l phenol red, and 0.1% BSA; pH adjusted to 7.2 with NaOH) as described previously (5). Media were collected in 1- or 5-min fractions and stored at -26°C. Hormone release also was measured by assaying the total hormone secreted (0.25 million cells/well) over 2 h of static incubation in testing medium under the culture conditions described above and in detail previously (5). BAPTA loading was accomplished during a 40-min wash period that preceded all experiments in static incubation conditions. GTH-II content of samples from both static incubation and cell-column perifusion experiments were determined by radioimmunoassay (38).

Hormone release measurements from static incubation studies were normalized to control cultures for each experiment and are therefore expressed as percentages of control (%control). For studies of hormone secretion in perifusion conditions, values were normalized to the mean of the first five measurements (i.e., before the first treatment) and are denoted as percentages of pretreatment (%pretreatment). Net hormone release responses of perifused cell columns to caffeine were quantified by integrating the area under the curve during the caffeine treatment (25 min). This was subdivided into "peak" response (first 10 min of caffeine treatment) and "plateau" response (last 15 min of caffeine treatment). Each time point was subtracted from the prepulse mean, defined as the average of the three time points before the caffeine challenge. Statistical analyses were performed with ANOVA, followed by Fisher's protected least significant difference post hoc test. Differences were considered significant when P < 0.05. Results are presented as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Caffeine-stimulated GTH-II release is Ca2+ dependent. Perifusion of dispersed goldfish pituitary cells with 10 mM caffeine resulted in a very large GTH-II release response (Fig. 1A). A second application of caffeine (45 min after the first) evoked only the plateau portion of the GTH-II release response (Fig. 1B). Significant GTH-II secretion was measured at 1 and 10 mM caffeine in static incubation experiments (Fig. 1C); 10 mM caffeine was used for the rest of the experiments. It is notable that caffeine is much less powerful in this assay of prolonged hormone release, suggesting the importance of a rapidly depleting or desensitizing component. Obvious candidates for such a "depletable" component are intracellular Ca2+ stores, which 10 mM caffeine is known to mobilize in many cell types.


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  		Fig. 1.   Characteristics of caffeine-stimulated gonadotropin (GTH-II) release. A: goldfish pituitary cells were treated with 10 mM caffeine (open horizontal bar) in a cell-column perifusion chamber. Samples were collected every 60 s to determine the kinetics of the GTH-II response (n = 6). B: sequential challenges with 10 mM caffeine (open horizontal bars) display desensitization (n = 4). Horizontal scale for B is the same as for A. All perifusion data are normalized to the pretreatment GTH-II levels in each column. Caff, caffeine. C: dose-response relationship of caffeine-stimulated GTH-II release was assessed in 2-h static incubation studies (n = 12). star Significant difference compared with control.

In accordance with the idea that caffeine acts by mobilizing intracellular Ca2+ stores, caffeine generated Ca2+ signals in single identified gonadotropes (Fig. 2A). Caffeine-stimulated Ca2+ signals and GTH-II release responses were abolished by loading cells with the high-affinity Ca2+ chelator BAPTA, accomplished by preincubation with 50 µM BAPTA-AM (Fig. 2C). Although the possibility of nonspecific BAPTA actions cannot be totally ruled out, these data suggest that caffeine-stimulated GTH-II release is not mediated by a parallel Ca2+-independent signaling cascade. In preliminary experiments, caffeine-stimulated GTH-II release was significantly inhibited when cells were subjected to prolonged nominally extracellular Ca2+-free conditions that would be expected to deplete intracellular Ca2+ pools by preventing capacitative Ca2+ entry (n = 3, data not shown).


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  		Fig. 2.   Caffeine-stimulated GTH-II release requires localized Ca2+ signals. A: temporal profile of Ca2+ signals generated by treatment of identified, fura 2-loaded gonadotropes with 10 mM caffeine (open horizontal bar). The trace is representative of 6 other gonadotropes from separate cultures. Caffeine-stimulated Ca2+ signals were not observed in 3 cells preincubated with 50 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM for 30 min. B: spatial characteristics of caffeine-evoked Ca2+ signals in a representative gonadotrope from the same data set as in A. Images are 15 s apart and are read from left to right, starting at the top. A cartoon of this gonadotrope is provided for reference. C: 10 mM caffeine-stimulated GTH-II (2-h static incubation) is abolished by preincubating cells in 50 µM BAPTA-AM (n = 20). star Significant difference compared with control.

Although acute caffeine-elicited GTH-II responses were severalfold greater than those induced by GnRH, Ca2+ signals evoked by caffeine were of similar maximal amplitude to those stimulated by sGnRH or cGnRH-II (24). Peak [Ca2+]i, measured over the bulk cytosol, rose only 157.5 ± 33.6 nM above baseline in the presence of 10 mM caffeine. It is important to note that the actual [Ca2+]i in some regions of the cell is likely misrepresented by these bulk measurements, because the peak of the caffeine-evoked Ca2+ signal was highly localized (Fig. 2B). In all cells tested, caffeine generated Ca2+ signals that were initially focused in the region opposite the nucleus, which is known to be rich in secretory granules (50), but subsequently spread across the cell. Collectively, these data suggest that caffeine-stimulated GTH-II release requires Ca2+ signals initiated by spatially localized Ca2+ stores.

Protein kinase A/cAMP, K+ channels, and VGCC do not mediate caffeine-stimulated GTH-II release. Xanthines, such as caffeine, have been shown to modulate the cAMP pathway through inhibition of phosphodiesterases. The actions of cAMP are mediated through H-89-sensitive protein kinase A (PKA) in goldfish gonadotropes (6). In the present study, we investigated whether actions on the cAMP/PKA system underlie the GTH-II-releasing effect of caffeine. Alone, H-89 (10 µM) reduced GTH-II release by ~50%, suggesting a role for the cAMP/PKA pathway in the maintenance of basal release in this cell type. However, H-89 had no significant effect on caffeine-evoked GTH-II release (Fig. 3). This result agrees with the observation that the massive hormone release response to caffeine is not mimicked, in previous experiments, by IBMX, a more specific phosphodiesterase inhibitor (8). Together, these data indicate that caffeine signaling in goldfish gonadotropes does not have a prominent cAMP/PKA component and that basal and agonist-evoked GTH-II release may be controlled independently.


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  		Fig. 3.   Caffeine-stimulated GTH-II release is not mediated by the cAMP/protein kinase A (PKA) pathway. Effects of 10 µM H-89 (filled horizontal bar)on GTH-II release evoked by 10 mM caffeine (open horizontal bar) are shown. Pooled results are shown (n = 6). Inset: quantified GTH-II release responses. Quantification was done as described in METHODS. Units are cumulative %pretreatment.

Caffeine has been reported to inhibit K+ conductances in cell lines derived from mammalian pituitary cells (see e.g., Ref. 1). To investigate this possibility, we recorded from identified gonadotropes in the whole cell configuration. As has been shown previously (48), depolarizing steps in the presence of TTX and Cd2+ evoked a biphasic outward K+ current (Fig. 4A). Caffeine treatment rapidly decreased the amplitude of both the peak and plateau portions of this current by ~40% in a voltage-independent and reversible manner (Fig. 4, B-E). Although these data indicate that caffeine blocks both the A current (IK(A)) and the delayed rectifier current, they do not show that the blockage of K+ currents mediates the secretagogue actions of caffeine. To evaluate this possibility, we examined whether caffeine-stimulated GTH-II release is sensitive to tetraethylammonium (TEA), a broad-specificity inhibitor of K+ currents in many cell types. In goldfish gonadotropes, 5 mM TEA is known to block the majority of the K+ current (48). Surprisingly, caffeine-stimulated GTH-II release was not reduced by 5 mM TEA (Fig. 5). These results indicate that although caffeine may modulate ionic currents in the plasma membrane, these events are dissociated from the signaling cascades that mediate caffeine stimulation of exocytosis.


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  		Fig. 4.   Caffeine inhibits K+ channels. Total outward currents recorded at different step pulses under control conditions (A) and in the presence of 10 mM caffeine (B) are shown. C: caffeine attenuates both the peak and plateau phases of outward currents. D: caffeine inhibition of K+ currents has a relatively fast onset and is reversible. Horizontal bars denote stages of drug perifusion. Because the bath volume is ~250 µl, complete changeover is expected in <1 min. E: caffeine inhibition of K+ channels is voltage independent. I/Imax, current normalized to the amplitude of the peak current under control conditions. Where examples are shown, data are representative of at least 3 independent trials.



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  		Fig. 5.   Caffeine-stimulated GTH-II release is not mediated by actions on K+ channels. A: effects of 5 mM TEA (horizontal filled bar) on GTH-II release evoked by 10 mM caffeine (horizontal open bar). B: quantified GTH-II responses (n = 6).

Although acute GnRH-stimulated GTH-II release evoked by the two GnRHs is independent of extracellular Ca2+ (23), influx through VGCCs is important in the maintenance of a prolonged secretory response, especially to cGnRH-II (7). Thus we have hypothesized a role for VGCC in the refilling of the rapidly exchanging Ca2+ pools sensitive to both GnRHs. Goldfish gonadotropes possess a single class of VGCCs, which are high-voltage activated, inactivation resistant, and completely blockable by micromolar Cd2+ (48). Blockade of VGCCs with 50 µM Cd2+ did not block either phase of the caffeine-evoked GTH-II release response (Fig. 6A), even when given up to 35 min before the application of caffeine (Fig. 6B), suggesting that caffeine-sensitive Ca2+ stores are not maintained through the activity of VGCCs. Treatment with 50 µM Cd2+ alone caused an increase in GTH-II release of variable amplitude (Fig. 6A). Like the large secretagogue response of gonadotropes when conditions are switched to Ca2+-free medium, we have previously attributed this to the homeostatic release of Ca2+ from intracellular stores (23).


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  		Fig. 6.   Caffeine-stimulated GTH-II release is mediated by depletion-resistant Ca2+ stores. A: GTH-II release evoked by 10 mM caffeine, with or without coapplication of 50 µM Cd2+ (horizontal hatched bar). Inset: quantified GTH-II responses. star Significant difference compared with control (n = 6). B: GTH-II response to 10 mM caffeine (horizontal open bar) also is not inhibited by prolonged pretreatments with 50 µM Cd2+ (shaded horizontal bars). Inset: quantified hormone release responses (n = 6).

Independence from typical RyR and SERCA-containing Ca2+ stores of the ER. The effects of 10 mM caffeine are most commonly attributed to the activation of RyR. In the present study, we used a high dose of ryanodine and a two-pulse protocol to test whether caffeine-stimulated GTH-II release involves RyR. This protocol was selected because ryanodine binds to the RyR in its open conformation (11, 43) and, therefore, may exert a block that is use dependent in some systems. Our results clearly demonstrate that 100 µM ryanodine has no effect on either caffeine pulse, compared with untreated controls (Fig. 7, A and B). The GTH-II response evoked by caffeine also was not inhibited by 50 µM dantrolene, another inhibitor of RyR (Fig. 7C). The potentiation of hormone release by dantrolene might have resulted from a shunting of Ca2+ into a caffeine-sensitive pool. Collectively, these data indicate that caffeine-stimulated GTH-II release is not mediated through activation of the RyRs, which are presumed to be components of ER Ca2+ stores (3, 41).


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  		Fig. 7.   Caffeine-stimulated GTH-II release does not require endoplasmic reticulum ryanodine receptors (RyR). A: treatment with 100 µM ryanodine (horizontal filled bar) does not inhibit sequential GTH-II responses elicited by caffeine (horizontal open bars). B: quantified data from A (n = 8). Ryano, ryanodine. C: caffeine-stimulated GTH-II release is not inhibited by 50 µM dantrolene (30-min pretreatment) in perifusion experiments similar in design to those shown in Fig. 3 (n = 8). Dantro, dantrolene. star Significant difference compared with control.

Another hallmark of typical agonist-sensitive ER Ca2+ stores is the activity of SERCA pumps, which refill ER Ca2+ pools in all cell types studied to date (41). Treatment with 10 µM BHQ, a concentration that blocks SERCA (33) and that inhibits the Ca2+ stores common to both GnRHs in goldfish gonadotropes (23), had no effect on caffeine-stimulated GTH-II release (Fig. 8). GTH-II release responses to caffeine also are not blocked by 2 µM thapsigargin (Johnson JD and Chang JP, unpublished results). Collectively, these findings strongly suggest that caffeine-sensitive Ca2+ stores that modulate GTH-II release may be largely independent of classic ER Ca2+-ATPases.


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  		Fig. 8.   Caffeine-stimulated GTH-II release does not require sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs). Effects of 10 µM 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ; horizontal filled bar) on GTH-II release evoked by 10 mM caffeine (horizontal open bar). Inset: quantified GTH-II responses (n = 8).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Relationship between caffeine- and sGnRH-sensitive Ca2+ stores. Caffeine-sensitive Ca2+ stores are highly coupled to GTH-II release in cultured goldfish gonadotropes. These Ca2+ stores also are of particular interest, because they represent a clear difference between the signaling cascades of the major endogenous GTH-II secretagogues, sGnRH and cGnRH-II. Acute GTH-II stimulation by both GnRHs depends on the mobilization of intracellular Ca2+ stores that are sensitive to TMB-8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)octoyl ester] and SERCA inhibitors. This common store seems to be highly sensitive to the manipulation of extracellular Ca2+ levels and probably requires VGCCs for refilling (23). Recent work from our laboratory (23) has shown that, between the two neuropeptides, only sGnRH signaling involves caffeine-sensitive mechanisms. This correlated well with the observation that GTH-II release evoked by sGnRH, but not cGnRH-II, requires an IP3-sensitive Ca2+ store that is resistant to depletion under conditions that reduce the availability of extracellular Ca2+ (6, 7, 19, 23). Caffeine has been reported to inhibit several aspects of the IP3 signaling system (11, 45). The Ca2+ dependence and the Cd2+ insensitivity of caffeine-stimulated GTH-II release, revealed in the present study, provide further evidence that caffeine may act on the same depletion-resistant Ca2+ store as sGnRH. The apparent slow Ca2+ exchange of the caffeine-sensitive Ca2+ pool may result from a lack of SERCA pumps (see Novel properties of caffeine-mediated Ca2+ release leading to exocytosis). The difference in magnitude between GTH-II release responses evoked by caffeine when compared with sGnRH can be explained if caffeine modulates multiple Ca2+ pools. Perhaps the secretagogue effects of caffeine are not manifested through the same Ca2+ stores that mediate the effects of caffeine on sGnRH signaling.

Features of caffeine-evoked Ca2+ signals. In the present study, experiments including the use of BAPTA suggest that the ability of caffeine to generate Ca2+ signals is essential for caffeine to stimulate GTH-II exocytosis. However, given the disparity in the magnitude of the relatively small global Ca2+ signals over the course of caffeine treatment and the large hormone release response, we speculate that caffeine evokes highly localized Ca2+ release from intracellular stores that are in close proximity to sites of GTH-II secretion. Consistent with this hypothesis, caffeine generated Ca2+ signals that began as highly localized events near a region of the membrane opposite the nucleus. Previous morphological studies have suggested that goldfish gonadotropes exhibit a weak polarity in culture and that this region opposite the nucleus is often rich in secretory granules and globules (50). The location of these events was similar in all cells tested, at least with respect to the ultrastructural landmarks (nucleus and globules) that are visible using DIC optics. One major advantage of our morphological identification protocol is that cells are selected for a specific orientation, thereby ensuring that they are imaged on roughly the same focal plane relative to the nucleus. However, beyond rough estimates of the localization of Ca2+ signals, the low spatial and temporal resolution of our data do not permit extrapolation to the level of Ca2+ microdomains, which are known to be important in the regulation of exocytosis and other localized cellular functions, in many cell types (3, 21, 35).

Novel properties of caffeine-mediated Ca2+ release leading to exocytosis. In rat gonadotropes, Tse et al. (47) have suggested that the strong coupling of GnRH signaling to GTH-II exocytosis results from the close proximity of agonist-sensitive ER stacks to secretory granules (and presumably exocytotic protein machinery). In the rat model, acute GnRH signaling follows a classic pathway and is mediated by a single ER Ca2+ pool that is IP3 sensitive, rapidly exchanging, and refilled by SERCA pumps (44, 46). Caffeine-sensitive Ca2+ stores, if present, have not been assigned a significant role in the generation of basal or GnRH-evoked Ca2+ signals in mammalian gonadotropes. However, a large body of work (6, 7, 24) has demonstrated many differences in GnRH signal transduction and, specifically, Ca2+ signaling between rats and goldfish. Indeed, the characteristics of caffeine-stimulated GTH-II release in the present study differ substantially from what we would expect of typical agonist-sensitive ER Ca2+ pools. First, on the basis of hormone release experiments with Cd2+, it appears that caffeine-sensitive Ca2+ stores may not be rapidly exchanging. Second, caffeine action is independent of RyR and SERCA pumps, which are known to mediate Ca2+ flux from caffeine-sensitive ER of many cell types (41). Caffeine-stimulated GTH-II release also is unlikely to be mediated by mitochondria, because it is not blocked by 10 µM carbonyl cyanide m-chlorophenylhydrazone (n = 4, data not shown), a mitochondrial uncoupler shown to modulate the shape of Ca2+ signals in mammalian gonadotropes (26). Interestingly, a recent report (18) indicated that the involvement of cyclopiazonic acid (CPA)-sensitive SERCA pumps in caffeine-evoked Ca2+ release may be tissue dependent. Thapsigargin-insensitive Ca2+ stores are found in many cell types, including several pituitary cell lines. However, these have not been described to be agonist or caffeine sensitive (40). Nevertheless, these features suggest the possible involvement of several classes of non-ER Ca2+ stores, including acidic compartments such as the secretory granules, in caffeine-stimulated GTH-II release and, by association, suggest a possible role in sGnRH signaling.

Given the localization of caffeine-evoked Ca2+ signals and the pharmacological evidence suggesting a role for non-ER Ca2+ stores, we speculate that caffeine may act on targets close to or on the secretory granules themselves. Secretory granules contain very large quantities of Ca2+. In several cell types, Ca2+ accumulation into granules is SERCA independent; instead they are filled by H+/Ca2+ exchange or possibly as a result of endocytosis (32, 37). The slow Ca2+ uptake was a primary reason why acidic organelles were rejected as candidates for the prototypical agonist-sensitive store, subsequently attributed to the ER (41). Nevertheless, there is growing evidence that the secretory granules may constitute a physiologically relevant IP3 (agonist)-sensitive Ca2+ store in a number of experimental systems (21, 37). Although the most unequivocal evidence has come from studies of exocrine cell types (13, 32, 36), there also is evidence of novel Ca2+ stores associated with the secretory granules in endocrine cells (25). Future work, including the direct measurement of Ca2+ levels inside the secretory granules of intact goldfish gonadotropes, is required to confirm this proposal.

The observation that caffeine action is ryanodine insensitive is unusual but not unprecedented. For example, the intracellular Ca2+ release component of Ca2+ transients in the presynaptic terminals of goldfish retinal bipolar cells is sensitive to caffeine but not to ryanodine (28). It has been proposed for mouse pancreatic islets that a caffeine-sensitive intracellular Ca2+ store modulated glucose-stimulated Ca2+ oscillations, an effect that was not mimicked by ryanodine (42). There also are several accounts in the literature of RyR that are caffeine insensitive (9, 14, 29). Further work, including direct measurements of Ca2+ within organelles, is required to characterize the putative novel class of Ca2+ stores in goldfish gonadotropes.

Differential coupling of signaling systems to GTH-II secretion. Another interesting conclusion from this study is that one part of the caffeine signal transduction circuit is dissociated from GTH-II release. Treatments with K+ channel blockers such as TEA or 4-aminopyridine (4-AP) are known to depolarize gonadotropes (Wong CJH and Change JP, unpublished observations), which would presumably activate VGCCs. Nonetheless, although caffeine inhibits K+ channels, caffeine-stimulated GTH-II release does not result from the inhibition of K+ channels, as indicated by the lack of effect of TEA. Similarly, caffeine-stimulated GTH-II release is not affected by 4-AP, an inhibitor of IK(A) in goldfish gonadotropes (48) (Wong CJH and Chang JP, unpublished observations). The reason for this uncoupling remains to be elucidated. Direct activation of VGCC with high KCl or BAY K 8644 generates Ca2+ signals (23) (Van Goor F and Chang JP, unpublished observations) and have been reported to stimulate GTH-II release (49). In contrast, TEA treatment failed to increase GTH-II release in the present study, suggesting that the site of this functional "uncoupling" may be situated downstream of K+ channels but upstream of VGCC. Accordingly, preliminary experiments have shown that 5 mM TEA evokes weak Ca2+ signals in single gonadotropes (Wong CJH and Chang JP, unpublished). Together, these data suggest the existence of selective uncoupling between events that modulate electrical excitability of the plasma membrane and hormone release in this cell type.

Comparison with goldfish somatotropes. The intracellular mechanisms mediating the control of hormone release by sGnRH and cGnRH-II differs dramatically between goldfish gonadotropes and somatotropes (6, 7). In somatotropes, growth hormone release evoked by both GnRHs is mediated by caffeine-sensitive Ca2+ stores that are largely SERCA independent (22). However, unlike the agonist-specific Ca2+ stores described in the present work, caffeine/GnRH-sensitive Ca2+ stores in somatotropes are rapidly depleting (<5 min), partially sensitive to ryanodine, and dependent on cAMP signaling (22, 52). Comparisons of the mechanisms of caffeine-stimulated hormone release between gonadotropes and somatotropes may lead to important insights into how these two closely related cell types are differentially controlled by neuroendocrine regulators, including the GnRHs.

Physiological relevance. In the present report, we have explored some pharmacological properties of signaling cascades linking an agonist-specific Ca2+ store to the control of hormone exocytosis. It is tempting to speculate that caffeine-sensitive Ca2+ stores may be involved in the differential control of gonadotrope function by sGnRH and cGnRH-II (27). Caffeine and sGnRH have similar seasonal profiles with respect to their maximum efficacy in releasing GTH-II in vitro, with both being most efficient in between the stages of gonadal recrudescence and maturation (20, 31). Compared with the kinetics of cGnRH-II-stimulated GTH-II release, responses to sGnRH become very extended during this period (31), raising the possibility that the prolonged component may be due to the recruitment of caffeine-sensitive intracellular Ca2+ stores. Ca2+ signals generated by sGnRH also have slower rates of rise and more often exhibit a prolonged monophasic waveform compared with those of cGnRH-II (24). Although it has been elegantly demonstrated in a few cell types (e.g., Ref. 10; reviewed in Ref. 21), the functional specificity of Ca2+ signals has not been demonstrated within single pituitary cells from any species. Despite the fact that multiple GnRHs have been discovered in species from all vertebrate classes and in man (51), the signal transduction of two GnRHs has been extensively compared only in goldfish. These comparative analyses are providing novel insights into the diversity of Ca2+ signaling systems.


    ACKNOWLEDGEMENTS

We acknowledge the support of the National Sciences and Engineering Research Council of Canada for operating grant OGP121399 to J. P. Chang, postdoctoral fellowships to C. J. H. Wong and J. D. Johnson, and a postgraduate studentship to W. K. Yunker. J. D. Johnson received support from a Province of Alberta Graduate Fellowship and the Andrew Stewart Memorial Prize.


    FOOTNOTES

* J. D. Johnson and C. J. H. Wong contributed equally to this work.

Present address of J. D. Johnson: 815 Yalem Bldg., Renal Division, Mailstop: 9032648, Washington University Medical Center, BJH North, 216 South Kingshighway Blvd., St. Louis, MO, 63110.

Address for reprint requests and other correspondence: J. P. Chang, Dept. of Biological Sciences, Biological Sciences Bldg., CW 405, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2E9 (E-mail: john.chang{at}ualberta.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00044.2001

Received 9 February 2001; accepted in final form 2 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 282(3):C635-C645
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society



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