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Wortmannin-Sensitive and -Insensitive Steps in Calcium-Controlled Exocytosis in Pituitary Gonadotrophs: Evidence That Myosin Light Chain Kinase Mediates Calcium-Dependent and Wortmannin-Sensitive Gonadotropin Secretion

Kang Rao, Won-Young Paik, Lixin Zheng, Richard M. Jobin, Melanija Tomi, He Jiang, Satoshi Nakanishi and Stanko S. Stojilkovic

Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. (S.N.), Tokyo, Japan

Address all correspondence and requests for reprints to: Dr. Stanko S. Stojilkovic, National Institute of Child Health and Human Development, Endocrinology and Reproduction Research Branch, Building 49, Room 6A-36, 49 Covent Drive, MSC 4510, Bethesda, Maryland 20892-4510. E-mail: stankos{at}helix.nih.gov

	Abstract

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In cultured rat pituitary cells, increases in the cytosolic calcium concentration ([Ca²⁺]_i) and LH release are induced by activation of GnRH receptors as well as by nonreceptor-mediated stimuli. Treatment of pituitary cells with the myosin light chain kinase (MLCK) inhibitor, wortmannin, attenuated GnRH-induced LH release. Wortmannin also reduced the LH responses to nonreceptor-mediated elevation of [Ca²⁺]_i by ionomycin and activation of voltage-sensitive Ca²⁺ channels by Bay K 8644 or high K⁺, as well as Ca²⁺-induced LH release in permeabilized pituitary cells. The [Ca²⁺]_i responses to these stimuli were unaltered in wortmannin-treated pituitary cells, indicating that this compound inhibits a Ca²⁺-dependent step in exocytosis without affecting Ca²⁺ signaling. In perifused pituitary cells, the GnRH-induced early spike phase of LH release was not affected by wortmannin, whereas the subsequent plateau phase was almost completely inhibited. No significant changes in GnRH-induced phospholipase D activity and diacylglycerol production were observed in wortmannin-treated pituitary cells during the sustained phase of agonist stimulation. Wortmannin also had no effect on LH responses to the protein kinase C activator, phorbol 12-myristate 13-acetate, further indicating that the attenuation of agonist-induced LH release is not related to inhibition of the diacylglycerol/protein kinase C pathway. In addition, agonist-induced LH release was attenuated by two other MLCK inhibitors, MS-347a and KT5926. These data suggest that MLCK mediates the downstream effects of Ca²⁺ on exocytosis, an action supported by the finding of wortmannin-sensitive phosphorylation of a 20-kDa protein in pituitary cells and T3–1 gonadotrophs treated with GnRH, K⁺, and Bay K 8644. This protein was coprecipitated from pituitary extracts with a specific antibody to nonmuscle myosin IIB and comigrated with 20-kDa smooth muscle myosin light chain on SDS-PAGE. These results demonstrate that Ca²⁺ controls exocytosis through an initial wortmannin-insensitive step and a sustained wortmannin-sensitive step and suggest that the latter event in the cascade of cellular responses is dependent on phosphorylation of nonmuscle myosin IIB light chain by MLCK.

	Introduction

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IN MANY secretory cells, calcium appears to be the major, if not the exclusive, messenger that is responsible for the control of exocytosis. Two hypotheses have been suggested to explain the manner in which elevations of the cytosolic calcium concentration ([Ca²⁺]_i) are coupled to exocytosis (1). The first of these proposes that specific proteins on the secretory vesicle membrane interact with sites on the inner surface of the plasma membrane in a calcium-dependent manner, and that elevation of [Ca²⁺]_i promotes this interaction and leads to exocytosis. The second hypothesis posits that fusion of secretory vesicles with the plasma membrane is prevented by elements of the cytoskeleton, and that increases in [Ca²⁺]_i cause fusion by disinhibition of this process. At present, little information is available to differentiate between these two proposed mechanisms for the control of exocytosis. It is also unclear whether Ca²⁺ controls exocytosis directly, by binding to cytoskeletal or secretory vesicle proteins that, in turn, activate exocytosis, or indirectly, by activation of multifunctional calcium-dependent protein kinases in the cytoplasm. The first model implies that any threshold rise in [Ca²⁺]_i will activate exocytosis. In the second model, Ca²⁺ serves only as a trigger in the cascade of cellular events leading to secretion. Two potential pathways for such multistep coupling between Ca²⁺- and calcium-dependent protein kinases are Ca²⁺protein kinase Cannexinsexocytosis and Ca²⁺calmodulinCa²⁺/calmodulin kinase IIsynapsin Iexocytosis (2).

In addition to activation of calmodulin-dependent kinase II, the Ca²⁺-calmodulin complex is involved in the control of myosin light chain kinases (MLCKs) and several other enzymes. MLCKs belong to a heterogeneous group of enzymes that are broadly classified into invertebrate striated muscle and vertebrate smooth muscle/nonmuscle types (3). In smooth muscle cells, Ca²⁺ released from the sarcoplasmic reticulum is bound by calmodulin to form a complex that subsequently binds to and activates MLCK. The activated kinase phosphorylates a specific serine residue in the regulatory light chain that contributes to the control of myosin’s interactions with actin. A decrease in [Ca²⁺]_i leads to dissociation of the kinase/calmodulin complex as well as isomerization of the kinase to its inactive form (4). In rat basophilic cells, Ca²⁺ has been proposed to act through calmodulin to activate MLCK, which, in turn, stimulates exocytosis (5). In these cells, secretion is associated with diphosphorylation of myosin light chains (MLCs) by MLCK as well as phosphorylation by protein kinase C. Selective inhibition of light chain phosphorylation by either kinase suppresses exocytosis, indicating that the coordinate actions of MLCK and protein kinase C are required for agonist-induced secretion (6). In accord with this, a rise in [Ca²⁺]_i alone was not sufficient to initiate exocytosis in RBL-2H3 basophilic cells (7). The participation of MLCK in exocytosis has also been implicated in studies with permeabilized chromaffin cells (8) and insulin-secreting HIT-T15 cells (9).

These observations have raised several questions about the importance of MLCKs in the control of exocytosis. 1) Does this process represent a common mechanism? 2) Is activation of protein kinase C necessary for such Ca²⁺-dependent and MLCK-mediated exocytosis? 3) In addition to Ca²⁺/MLCKs, what other signaling molecules are required for Ca²⁺-dependent exocytosis? These and other questions were addressed in cultured rat pituitary cells in which, in contrast to RBL-2H3 cells, nonreceptor-induced elevations in [Ca²⁺]_i are sufficient to stimulate hormone release from normal and immortalized gonadotrophs and other secretory cell types (10, 11, 12). Pituitary gonadotrophs are normally regulated by Ca²⁺-mobilizing GnRH receptors (13), but also release LH in response to activation of protein kinase C by phorbol esters (14, 15). The present findings demonstrate that Ca²⁺ can promote exocytosis in gonadotrophs through a cascade of cellular events that includes activation of MLCK during the sustained, but not the initial, phase of secretion.

	Materials and Methods

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Chemicals
MLCK inhibitors were provided by Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. (Tokyo, Japan), and U73122 and U73433 were supplied by Dr. J. E. Bleasdale, Upjohn Co. (Kalamazoo, MI). GnRH was obtained from Peninsula Laboratories (Belmont, CA), and indo-1/AM was purchased from Molecular Probes (Eugene, OR). [³H]Oleic acid, [³H]cytidine, and Econofluor-2 were obtained from DuPont-New England Nuclear (Boston, MA). Myo-[³H]inositol was purchased from Amersham (Arlington Heights, IL). [³²P]Orthophosphoric acid was obtained from DuPont-New England Nuclear Research Products; Pansorbin was obtained from Calbiochem. Myosin IIA and IIB antibodies against specific peptide sequence at the C-terminal were provided by Dr. Robert S. Adelstein, Laboratory of Molecular Cardiology, NHLBI (16); okadaic acid was obtained from LC Laboratory (Woburn, MA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Cytosolic calcium measurements
For cytosolic calcium concentration measurements, cells (10⁶/dish) were plated on coverslips coated with poly-L-lysine and cultured in medium 199 containing Earle’s salts, sodium bicarbonate, 10% horse serum, and antibiotics. The next day, the cells were incubated at 37 C for 60 min with 2 µM fura-2/AM. The extracellular buffer used in Ca²⁺ measurements was phenol red-free medium 199 with Hanks’ salts or modified Krebs-Ringer buffer without Mg²⁺. Coverslips with cells were washed with phenol red-free buffer and mounted on the stage of an Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany) attached to the Attofluor Digital Fluorescence Microscopy System (Atto Instruments, Rockville, MD). Cells were examined under a x40 oil immersion objective during exposure to alternating 340- and 380-nm light beams, and the intensity of light emission at 520 nm was measured. In this way, light intensities and their ratio, F(340)/F(380), which reflects changes in the Ca²⁺ concentration, were followed in several single cells simultaneously.

[³H]Inositol labeling and stimulation of pituitary cells
Pituitary cells were loaded with 5 µCi myo-[³H]inositol for 24 h, then washed three times with inositol-free medium 199-Hanks’ Balanced Salt Solution containing 25 mM HEPES (pH 7.4) and 1% FBS and stimulated at 37 C. Reactions were terminated by the addition of 0.2 ml ice-cold perchloric acid (10%, vol/vol), and the dishes were placed on ice for 30 min before the cells were removed from each well by scraping. The cell suspensions were transferred into glass tubes and placed on dry ice for 30 min. After thawing, the suspensions were centrifuged at 4 C (800 x g for 15 min), and the supernatants were extracted in conical tubes containing 350 µl of a mixture of 1,1,2-trichlorotrifluoroethane and tri-n-octylamine (1:1, vol/vol) and 100 µl 10 mM EDTA. The tubes were vortexed for 3 min and centrifuged for 5 min at 400 x g, and the upper phases were transferred into Eppendorf tubes containing 2 µl phenol red. After neutralization, samples were applied to a HPLC column, and inositol phosphates were eluted with a linear gradient of ammonium phosphate (1% increase/min), as described previously (17). The radioactivity of the effluent was continuously monitored by an on-line radioactive flow detector.

Diacylglycerol (DG) measurements
Cells were cultured in four-well dishes for 2–3 days as described above, and [³H] cytidine diphosphate (CDP)-DG formation was detected by a modification of the method of Godfrey and Watson (18). Cells were incubated in 0.45 ml DMEM containing 0.1% BSA, L-glutamine, high glucose (4.5 g/liter), and NaHCO₃ (1.4 g/liter) at 37 C for 60 min with [³H]cytidine (5 µCi/ml). Fifty microliters of 100 mM LiCl were then added, followed 10 min later by 55 µl medium with agonist. Incubations were continued for up to 60 min and stopped by the addition of 0.5 ml dry ice-cold methanol. Cells were scraped from the plates, and lipids were extracted by vigorous vortexing with 2.75 ml chloroform-methanol-water (1:2:0.75, vol/vol/vol). After mixing with chloroform (1 ml) and water (1 ml), the samples were centrifuged at 800 x g for 10 min. The lower phase was then transferred into a new set of 15-ml tubes and washed with 4 ml methanol-1 N HCl (1:1, vol/vol). Aliquots of the lipid phases containing [³H]CDP-DG were dried under nitrogen and analyzed by liquid scintillation spectrometry in Econofluor-2 (DuPont/NEN, Wilmington, DE).

Measurement of phosphatidylethanol production
Two days after cell preparation, the culture medium in 35-mm culture dishes was changed to 1.1 ml DMEM containing 0.1% fatty acid-free BSA, L-glutamine, high glucose (4.5 g/liter), NaHCO₃ (1.4 g/liter), and 5 µM [³H]oleic acid. After 16- to 24-h incubation, stimuli were added to the culture dishes in 120 µl of the above medium in the presence or absence of 0.5% ethanol for the indicated times. Treatments were terminated by placing the dishes on ice, followed by removal of the medium and rinsing the dishes with 1 ml ice-cold saline. Phosphatidic acid (PA) and phosphatidylethanol (PEt) were measured as previously described (17).

Phosphorylation of MLC
Cells were cultured in 100-mm culture dishes at a density of 4 x 10⁶ and labeled with [³²P]orthophosphoric acid (100 µCi/ml) in 4 ml buffer A (119 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl₂, 1 mM CaCl₂, 0.1% BSA, and 25 mM piperazine-N,N'-bis [2-ethanesulfonic acid] NaOH, pH 7.2) for 2 h at 37 C. After being washed twice with 3 ml buffer A, the cells were treated with GnRH, 50 mM KCl, or 100 nM Bay K 8644 for 2–20 min at 37 C. In experiments with wortmannin and okadaic acid, the compounds were added 5 min before treatment with GnRH, KCl, or Bay K 8644. The reaction was stopped by aspirating the stimulating buffer, and dishes were immediately placed on ice. Myosin IIB was then purified by immunoprecipitation as described previously (19). After analyzing the purified myosin by gel electrophoresis, the gels were air-dried, exposed to a Kodak XAR x-ray film (Eastman Kodak, Rochester, NY), and developed using a Kodak X-Omat M20 processor. Gel segments corresponding to a standard 20-kDa phosphorylated smooth muscle light chain run on the same gel as a marker were excised, and phosphorylated MLCs were eluted in 950 µl NH₄HCO₃ (100 mM; pH 8). The eluate was then digested with 50 µl trypsin/L-p-tosylamino-2-phenylethyl chloromethyl ketone for at least 18 h in a shaking water bath at 37 C. The supernatants containing digested peptides were lyophilized, and the dried residues were dissolved in 30-µl aliquots of isoelectric focusing gel sample buffer (1.08 g urea; 180 µl ampholytes, pH 4–6; and 50 µl ß-mercaptoethanol, dissolved in water up to 3 ml). One-dimensional isoelectric focusing gel electrophoresis was conducted as previously described (6) at 1000 V for 1 h to separate the tryptic peptides. The gel was then dried on a heated vacuum gel dryer for 1 h and subjected to autoradiography for detection of phosphopeptides. Relative quantification of the phosphopeptides was conducted by scanning the exposed films with a densitometer (Molecular Dynamics, Sunnyvale, CA). Previous studies have shown that a constant amount of tryptic peptide (phosphorylated at Ser¹⁹) was recovered from unstimulated cells (20), and this was used as an internal standard. The amounts of all ³²P-labeled peptides were expressed as a percentage of the internal standard.

Secretory responses
For static cultures, dispersed pituitary cells were seeded at 0.5 x 10⁶ cells/well in 24-well plates (Falcon, Oxnard, CA). After 2 days, the incubation medium was replaced by warmed Hanks’ medium 199 with HEPES, containing selected concentrations of stimulants and inhibitors. After incubation for 3 h at 37 C in a water-saturated atmosphere of 5% CO₂ in air, 0.7 ml medium was carefully aspirated from each well and kept frozen. In parallel experiments, 2-day-old cultures of pituitary cells were permeabilized as previously described (21), using 1 U/ml streptolysin O and 1 mM ATP. The permeabilized cells were stimulated with GnRH or increased Ca²⁺ for 12 min at 37 C. Wortmannin was added to the extracellular, intracellular, and stimulation buffers, giving the cells a 25-min pretreatment with the compound before stimulation with calcium and GnRH. Column perifusions were performed on 3-day cultured cells using previously reported conditions (22). Briefly, 2 x 10⁷ cells were incubated with preswollen Cytodex-1 beads in 60-mm culture dishes and perifused with Hanks’ medium 199 containing 20 mM HEPES and 0.05% BSA for 60 min at a flow rate of 0.6 ml/min. After agonist stimulation, fractions were collected every min and stored at -20 C. LH content was assayed by RIA using the reagents and standard provided by the National Pituitary Agency (Baltimore, MD).

Calculations
Secretory data are presented as the mean ± SE. The significance of differences between means was derived by Student’s t test. In Ca²⁺ measurements, the traces shown are representative of experiments performed with similar results in at least three different batches of pituitary cells. The curves in all figures and their IC₅₀s were generated by a Macintosh computer (Apple Computer, Cupertino, CA) using a KaleidaGraph program (Synergy Software, Reading, PA).

	Results

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In pituitary gonadotrophs, activation of GnRH receptors stimulates phosphoinositide hydrolysis and the production of inositol 1,4,5-trisphosphate (InsP₃) and DG, leading to an oscillatory intracellular Ca²⁺ response and gonadotropin secretion (23). The phospholipase D pathway is also integrated into the cascade of receptor-mediated intracellular signaling through a protein kinase C-dependent mechanism (17). Both Ca²⁺ and protein kinase C have been implicated in agonist-induced LH release (23). As shown in Fig. 1A, GnRH (100 nM) induced a several-fold increase in LH release from static cultures of pituitary cells stimulated for 3 h. Wortmannin, a MLCK inhibitor produced by the fungus Talaromyces wortmannii KY12420 (24), inhibited agonist-induced gonadotropin release in a concentration-dependent manner with an IC₅₀ of 2 µM. Similar results were obtained for FSH measurements (not shown). In GnRH-stimulated cells, the other inhibitor of MLCK, MS-347a, but not the inactive compound MS-347b, also inhibited agonist-induced LH release with an IC₅₀ of 0.7 µM (Fig. 1B). KT5926, another inhibitor of MLCK, significantly attenuated GnRH-induced LH secretion in static cultures of pituitary cells with an IC₅₀ of 5 µM (not shown). None of these inhibitors completely abolished GnRH-induced LH release, suggesting the existence of a wortmannin-insensitive step in this process.

View larger version (20K): [in this window] [in a new window]   Figure 1. Concentration-dependent attenuation of agonist-stimulated LH release in static cultures by inhibitors of MLCK. A, Inhibition of GnRH-induced LH release by wortmannin. Data points are the mean ± SE of results from five experiments, each performed in sextuplicate. *, P < 0.01 vs. GnRH alone. B, Effects of MS-347a and MS-347b on GnRH (100 nM)-induced LH responses. Data points are the mean ± SE of sextuplicate determinations. *, P < 0.01 vs. GnRH alone. All compounds were dissolved in DMSO (final concentration, 0.05% or less). Basal release was examined in the absence and presence of 10 µM MS-347a.

View larger version (47K): [in this window] [in a new window]   Figure 2. Inhibition of GnRH-induced gonadotropin release from perifused pituitary cells by wortmannin. A, Control; B, wortmannin-treated cells. In both columns, cells were initially exposed to 10 nM GnRH for 15 min, and subsequently to 10 nM GnRH pulses for 10 min, followed by 30-min washing periods. The horizontal line indicates the duration of exposure to wortmannin. The results shown are representative of four experiments, each performed in duplicate.

In these cells, wortmannin reduced the inositol phosphate responses to GnRH (100 nM for 15 min) in a concentration-dependent manner, with an IC₅₀ of 4.5 µM (Fig. 3, left panels). However, the suppression of InsP₃ production was not complete at 10 µM wortmannin. Also, no obvious change in the InsP₃ response was observed within 5 min of stimulation with GnRH in wortmannin-treated cells, a time sufficient to almost completely inhibit GnRH-induced LH release (Fig. 2). Furthermore, activation of Ca²⁺ signaling by GnRH in T3–1 gonadotrophs was not significantly altered by wortmannin treatment (not shown). Similar results were obtained in cultured pituitary cells. After incubation with 10 µM wortmannin for 15 min, the oscillatory Ca²⁺ response to 0.1 nM GnRH (Fig. 3A) and the nonoscillatory peak and plateau responses to 100 nM GnRH (Fig. 3B) were similar in dimethylsulfoxide (DMSO)-treated (controls, left panels) and wortmannin-treated cells (right panels). There was no statistical difference in the frequency (A) or amplitude of spiking (B) in controls and WT-treated cells. However, there was a significant delay in the onset of the oscillatory response to GnRH in wortmannin-treated cells (see Fig. 3).

View larger version (25K): [in this window] [in a new window]   Figure 3. Effects of wortmannin on inositol phosphate and calcium signaling in pituitary gonadotrophs. Left panels, Concentration-dependent attenuation of Ins(1,3,4)P3 and Ins(1,4,5)P3 responses to GnRH in wortmannin-treated T3–1 gonadotrophs. Vertical dotted lines represent IC50 values. *, P < 0.01 vs. GnRH alone. Right panels, GnRH-induced calcium responses in single pituitary gonadotrophs. A and B, Cells were treated with 10 µM wortmannin for 15 min (right panels) or with 0.1% DMSO (left panels) and stimulated with 0.1 nM (A) and 100 nM (B) GnRH. A, Frequency of spiking (minutes-1); controls, 9 ± 2 (n = 16); WT-treated, 7 ± 1 (n = 14). Latency (seconds): controls, 13 ± 1 (n = 21); WT-treated, 18 ± 2 (n = 21; P < 0.01). B, Amplitude [ratio F(340)/F(380)]: controls, 1.7 ± 0.14 (n = 8); WT-treated, 1.6 ± 0.2 (n = 7). C, Cells were treated with 10 µM wortmannin (right) and DMSO (left) for 90–120 min, and then stimulated with 100 nM GnRH. Before recording, cells were stimulated with 100 nM GnRH for 30 min and washed. GnRH (100 nM) was applied again 30–60 min after washing the cells.

To evaluate more directly the participation of MLCK in the cascade of the intracellular signaling triggered by GnRH, the presence and phosphorylation of myosin in pituitary cells were evaluated. Preliminary immunoblotting studies with specific antimyosin sera demonstrated that nonmuscle myosin IIB is the major isoform present in pituitary cells and T3–1 gonadotrophs, which also contain about 10% myosin IIA and 15% myosin VII (not shown). Furthermore, GnRH caused time-dependent phosphorylation of a 20-kDa protein in both T3–1 gonadotrophs and pituitary cells. Autoradiographs of 20-kDa protein phosphorylation in T3–1 gonadotrophs and the smooth muscle MLC (20 kDa; MLC₂₀) standard are shown in Fig. 4, upper panels. Agonist-induced phosphorylation of MLC₂₀ was inhibited by wortmannin, with an IC₅₀ comparable to that observed in secretory studies. GnRH also induced an elevation of the 20-kDa MLC phosphorylation in pituitary cells, comparable to that observed in T3–1 gonadotrophs (Fig. 5A). Figure 5 also illustrates the effects of MLCK on phosphorylation of the 20-kDa smooth muscle standard.

View larger version (23K): [in this window] [in a new window]   Figure 4. Phosphorylation of MLC in T3–1 gonadotrophs. A, Time course of GnRH (100 nM)-induced phosphorylation of MLC20. B, Concentration-dependent inhibition of GnRH-induced MLC20 phosphorylation by wortmannin. SMLC, Smooth MLC20 standard; WT, wortmannin. In this and the following figures, phosphorylation of MLC20 is expressed in relative densitometric units.

View larger version (27K): [in this window] [in a new window]   Figure 5. Characterization of GnRH-induced phosphorylation of MLC20. A, Upper panel, Effects of GnRH (100 nM) on MLC20 phosphorylation in T3–1 gonadotrophs at 10 and 15 min and in pituitary cells at 10 min. SMLC, Phosphorylation of MLC standard by MLCK. A, Lower panel, MLC20 phosphorylation in GnRH-stimulated T3–1 cells in the presence of okadaic acid (10 µM). B, Effects of GnRH and okadaic acid (O.A.) on MLC20 phosphorylation in T3–1 cells.

Further studies of Ca²⁺-dependent exocytosis were performed in the absence of receptor-mediated signaling responses to isolate the distal steps of the secretory pathway. As noted above, gonadotropin release is stimulated by increases in [Ca²⁺]_i due to activation of Ca²⁺ entry through voltage-sensitive calcium channels by high K⁺ or the calcium channel agonist, Bay K 8644 (10), and by release of stored Ca²⁺ by ionophores (30). Although less effective than GnRH, treatment with high K⁺, Bay K 8644, and ionomycin caused substantial increases in LH secretion from cultured pituitary cells (Figs. 6 and 7). It is well established that the rises in [Ca²⁺]_i induced by high K⁺, ionomycin (Fig. 8), and Bay K 8644 (10, 23) are responsible for their secretory actions. In analyzing the mechanism(s) of Ca²⁺-dependent exocytosis, we first excluded the participation of phospholipase C, phospholipase D, and some of the protein kinase C isozymes, which in some cell types are activated by elevation of cytosolic calcium (31, 32, 33). However, no changes in the products of phospholipase C and phospholipase D activity were observed during exposure of pituitary cells or T3–1 gonadotrophs to high K⁺ (Table 1). In addition, the secretory response to Bay K 8644 was not altered in protein kinase C-depleted cells (Fig. 9A). These results further support the hypothesis that nonreceptor-mediated rises in [Ca²⁺]_i are not associated with activation of these enzymes, and that the increase in [Ca²⁺]_i is directly responsible for LH release.

View larger version (23K): [in this window] [in a new window]   Figure 6. Inhibition of calcium-dependent LH release by wortmannin. A, Effects of wortmannin on Bay K 8644- and ionomycin-induced LH responses in intact pituitary cells. Bay K 8644 and ionomycin were dissolved in DMSO (final concentration, 0.1%). Wortmannin was dissolved in DMSO (final concentration 0.05% or less). Data points are the mean ± SE of sextuplicate determinations after 3-h incubation. #, P < 0.01 vs. control (Bay K 8644 and ionomycin without wortmannin). *, P < 0.01 vs. basal release. B, Effects of wortmannin on basal and GnRH-induced gonadotropin secretion in permeabilized pituitary cells. Cells were permeabilized by streptolysin O and bathed in medium containing the indicated concentrations of calcium for 12 min at 37 C. *, P < 0.01 between the same groups of cells with or without wortmannin. #, P < 0.01 between nontreated (basal) and treated cells.

View larger version (22K): [in this window] [in a new window]   Figure 7. Effects of wortmannin on depolarization-induced LH release from pituitary cells. A, Upper panel, Concentration-dependent effect of potassium on LH release in the presence and absence of wortmannin. A, Lower panel, Concentration-dependent inhibitory action of wortmannin on potassium-induced LH response. B, Upper panel, Concentration-dependent effect of extracellular potassium on MLC20 phosphorylation in T3–1 gonadotrophs stimulated for 10 min. Lower panel, Inhibitory effects of wortmannin on potassium- and Bay K 8644-induced MLC20 phosphorylation. *, P < 0.01 between the same groups of cells with or without wortmannin. #, P < 0.01 between nontreated (basal) and treated cells.

View larger version (17K): [in this window] [in a new window]   Figure 8. Lack of effect of wortmannin (WT) on depolarization- and ionomycin-induced calcium responses in single pituitary gonadotrophs. Cells were treated with 10 µM WT or DMSO (controls) for 15 min and subsequently with high potassium or ionomycin. The tracings shown are representative of at least six recordings for each treatment [amplitude, ratio F(340)/F(380): KCl, 0.4 ± 0.1; KCl plus WT, 0.4 ± 0.2; ionomycin, 1.2 ± 0.3; ionomycin plus WT, 1.5 ± 0.2].

View this table: [in this window] [in a new window]   Table 1. Lack of effect of wortmannin on CDP-DG, PEt, and PA production in GnRH- and PDBu-stimulated T3-1 gonadotrophs

View larger version (27K): [in this window] [in a new window]   Figure 9. Protein kinase C independence of wortmannin action on gonadotropin secretion. A, Bay K 8644-induced LH release in protein kinase C-replete and -depleted cells. LH release was stimulated by Bay K 8644 at concentrations of 1–100 nM (P < 0.05). No significant difference was observed between protein kinase C-replete and -depleted cells. Depletion of protein kinase C was induced by treatment with 1 µM PMA at room temperature for 24 h as previously described (22). Control cells were treated with 0.1% DMSO. B, Lack of effect of wortmannin on phorbol ester-induced LH responses. PMA was dissolved in DMSO (final dilution, 0.01%). *, P < 0.01 vs. basal.

In intact cells, the stimulatory effect of K⁺-induced depolarization on LH secretion was also inhibited in the presence of 10 µM wortmannin at all K⁺ concentrations examined (Fig. 7A, upper panel). In cells stimulated with 75 mM KCl, wortmannin inhibited LH release in a concentration-dependent manner, with an IC₅₀ of 2 µM (Fig. 7A, lower panel). A concentration-dependent increase in MLC₂₀ phosphorylation was observed in cells depolarized by high external potassium (Fig. 7B, upper panel). KCl- and Bay 8644-induced phosphorylation was reduced in cells exposed to wortmannin for 5 min (Fig. 7B, bottom panel). To examine the possible effects of wortmannin on intracellular Ca²⁺ levels in such treated cells, single gonadotrophs were stimulated with high K⁺ and ionomycin. As shown in Fig. 8, the cytosolic Ca²⁺ responses to 50 mM K⁺ and 5 µM ionomycin were unaffected by exposure to 10 µM wortmannin for periods ranging from 15–45 min (not shown). These data and the results of experiments with permeabilized cells demonstrate that the inhibitory action of wortmannin on calcium-induced LH release is not related to changes in [Ca²⁺]_i. They also indicate that the [Ca²⁺]_i changes in the submicromolar concentration range are sufficient to induce wortmannin-sensitive phosphorylation of MLC₂₀.

The possible effects of wortmannin on DG formation and protein kinase C-dependent steps in exocytosis were also evaluated. As shown in Table 1, there was no obvious difference in the CDP-DG, PEt, and PA responses of GnRH-stimulated gonadotrophs in the presence of wortmannin. The lack of effect of wortmannin on these responses shows that it does not affect the agonist-activated phospholipase D pathway. These results also indicate that a partial inhibition of phospholipase C activity does not affect integration of the phospholipase D pathway into the signaling response (17). In addition, no evidence for a direct effect of wortmannin on protein kinase C was obtained in cells stimulated by phorbol 12-myristate 13-acetate (PMA). As shown in Fig. 9B, PMA induced a concentration-dependent increase in LH release in pituitary cells bathed in Ca²⁺-deficient medium. This action of PMA was not associated with measurable changes in [Ca²⁺]_i (not shown), indicating that mobilization of intracellular Ca²⁺ stores is not responsible for its stimulation of LH release. Furthermore, wortmannin (10 µM) did not inhibit PMA-induced LH release at all concentrations studied (Fig. 9). These observations indicate that the inhibitory action of wortmannin on LH release is not related to changes in the agonist-induced messengers required for activation of protein kinase C or to inhibition of protein kinase C per se. They also demonstrate that the inhibitory action of wortmannin on LH release is specific for Ca²⁺-dependent mechanisms of secretion.

	Discussion

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Calcium has been suggested to control exocytosis either directly, by binding to the cytoskeletal and/or secretory vesicle proteins, or indirectly, through multistep actions that include calmodulin- and/or kinase-dependent mechanism(s) (1, 34, 35, 36). The first model implies that any threshold rise in [Ca²⁺]_i will activate exocytosis. In the second, rises in [Ca²⁺]_i should be dissociable from exocytosis by Ca²⁺-independent activation or inhibition of protein kinases. In accord with the latter proposal, the present data demonstrate that Ca²⁺ signaling and exocytosis can be pharmacologically dissociated in depolarized cells, GnRH-stimulated gonadotrophs, as well as permeabilized cells clamped at different calcium concentrations. In all cases, wortmannin inhibits LH release without affecting [Ca²⁺]_i. However, this inhibition is incomplete, suggesting the existence of both direct and indirect actions of Ca²⁺ on gonadotropin secretion.

In GnRH-stimulated cells, the initial phase (the first 2–4 min of stimulation) was not affected by wortmannin, whereas the sustained phase of secretion was dramatically reduced. Calcium signaling during these two phases differs in two respects: the sources of Ca²⁺ and the amplitude of the responses. The initial phase of Ca²⁺ spiking is dependent on Ca²⁺ mobilization, and the amplitudes of transients are in a low micromolar range. In contrast, the sustained phase of spiking is dependent on Ca²⁺ entry through voltage-gated calcium channels, and the amplitudes of these transients are in the submicromolar concentration range (23). The existence of a wortmannin-insensitive phase in depolarization-induced secretion indicates that it is not the source of Ca²⁺, but, rather, the amplitude of the [Ca²⁺]_i response that makes the critical difference in the direct and indirect actions of Ca²⁺ on secretion. Based on these observations, we may speculate that Ca²⁺ can activate exocytosis directly only when the localized [Ca²⁺]_i is high, and that such a microenvironment is reached in GnRH-stimulated cells by the mobilization of calcium from intracellular stores during the initial phase. In agreement with this, it has been shown recently that localized elevations of high [Ca²⁺]_i are needed in close proximity to the secretory vesicles to activate exocytosis in gonadotrophs by InsP₃, and that colocalization of the exocytotic sites and InsP₃ receptor channels provides the mechanism for such an effect (37).

In general, the indirect, wortmannin-sensitive action of calcium could be mediated by calmodulin and/or protein kinase C-dependent steps in hormone releases. As a rise in [Ca²⁺]_i is required for activation of several members of the protein kinase C family (38), this provides a potential mode for the coagonist actions of the two intracellular messengers on exocytosis. However, the secretory action of GnRH in pituitary cells is reduced (22), but not abolished, in protein kinase C-depleted cells (15, 22), indicating that Ca²⁺ also acts independently in exocytosis. In addition, PMA-induced LH release is not affected by wortmannin, further suggesting that the wortmannin-sensitive step in gonadotropin release is controlled by a distinct pathway.

Wortmannin was initially defined as an inhibitor of MLCK, with activity in the micromolar dose range (24). In addition to MLCK, wortmannin inhibits the activities of several other enzymes involved in the control of intracellular messenger pathways. For example, the activity of phosphatidylinositol 3-kinase, an essential component of the signal transduction pathways by tyrosine kinase receptors and several oncogene products, is inhibited by nanomolar concentrations of wortmannin (25, 26). However, it is unlikely that inhibition of this enzyme is relevant to the control of calcium-dependent exocytosis in gonadotrophs. In adrenal glomerulosa cells, wortmannin in micromolar concentrations attenuates the increases in both InsP₃ production and calcium signaling (39) due to its inhibition of phosphatidylinositol 4-kinase (27). In addition to this action on inositol phosphate signaling, wortmannin has been reported to cause inhibition of phospholipase D (28). Three lines of evidence indicate that the inhibitory action of wortmannin on LH exocytosis in pituitary gonadotrophs is not mediated by changes in the production of intracellular messengers, but, rather, by its action on a Ca²⁺-sensitive step in secretion.

First, GnRH-induced production of PEt, a marker for phospholipase D activity (17, 40), was unchanged during wortmannin treatment for 90 min. Likewise, no inhibition of CDP-DG formation by wortmannin was observed in GnRH-stimulated gonadotrophs, making it unlikely that protein kinase C or a protein kinase C-dependent step in exocytosis is affected. Wortmannin had no effect on PMA-induced LH release, as well. Second, although wortmannin reduced the InsP₃ response to GnRH, this did not prevent the initiation and reinitiation of GnRH-induced Ca²⁺ signaling in gonadotrophs. This is in accord with previous observations that ongoing Ca²⁺ spiking in agonist-stimulated gonadotrophs is relatively independent of changes in the InsP₃ concentration (41). Experiments with ionomycin, high K⁺, and Bay K 8644 also showed that the wortmannin-sensitive step in regulated exocytosis from pituitary cells is not related to calcium actions on signal-generating enzymes, as nonreceptor-mediated Ca²⁺ and LH responses were not associated with activation of the phospholipase C and phospholipase D pathways. Third, two other inhibitors of MLCK (42), KT5926 and MS-347a, but not the inactive compound MS-347b, inhibited GnRH-induced LH release in a manner comparable to that observed in wortmannin-treated cells.

Although the function of MLCK is well defined in smooth muscle cell contraction (4), little is known about the possible role(s) of this enzyme in secretory cells. By analogy with muscle cells, the inhibitory action of wortmannin on GnRH-stimulated LH release suggests that MLCK participates in exocytosis through the phosphorylation of cytoskeletal elements, specifically the MLC. Indeed, our experiments have demonstrated the presence of a nonmuscle myosin IIB in both pituitary cells and T3–1 gonadotrophs as the major isoform, and myosin IIA and myosin VII as the minor forms. Furthermore, the receptor and nonreceptor-mediated increases in [Ca²⁺]_i were associated with phosphorylation of a wortmannin-sensitive MLC₂₀. Inhibition of endogenous phosphatases by okadaic acid was followed by a several-fold increase in MLC₂₀ phosphorylation, and this was further enhanced in the presence of GnRH. Thus, pituitary cells express a functional calmodulin-MLCK-phosphatase system for the control of phosphorylation of MLC₂₀ in a manner comparable to that observed in smooth muscle cells.

In addition to gonadotrophs, the importance of cytoskeletal elements in exocytosis has been noted in several studies, and MLCK has been proposed to participate in receptor-activated exocytosis in chromaffin cells, basophilic RLB-2H3 cells, and insulin-secreting HIT-T15 cells (5, 6, 8, 9). In RLB-2H3 cells, MLC was phosphorylated by MLCK at threonine 18 and serine 19, and also by protein kinase C at serine 1 or 2, suggesting a requirement for both kinases in exocytosis (6). In accord with this, Ca²⁺ and phorbol ester (PMA) alone do not initiate exocytosis, but in combination are potent stimuli of secretion in these cells (7). That is not the case with gonadotrophs, in which a rise in [Ca²⁺]_i alone is sufficient to induce LH release. Also, Bay K 8644-induced LH responses were not affected in pituitary cells depleted of protein kinase C, further supporting the hypothesis that activation of the calcium-dependent and wortmannin-sensitive pathway is sufficient to induce LH release, albeit of a smaller magnitude than the agonist-induced response.

In conclusion, these data provide evidence that both receptor- and nonreceptor-induced rises in [Ca²⁺]_i activate exocytosis in pituitary gonadotrophs, and that this effect is not a consequence of indirect actions of calcium on the phospholipase C or phospholipase D signaling pathways. The data further demonstrate that Ca²⁺ signaling and LH secretion can be uncoupled by wortmannin in depolarized cells during the sustained, but not the initial, agonist stimulation. These observations in GnRH-stimulated cells suggest that Ca²⁺ controls exocytosis through an initial wortmannin-insensitive step and a sustained wortmannin-sensitive step. The sensitivity of Ca²⁺-dependent exocytosis to several other MLCK inhibitors strongly supports a model in which Ca²⁺ binds to calmodulin, leading to activation of MLC and control of sustained secretion. In fact, pituitary cells express several forms of nonmuscle myosins, including MLC₂₀, and a MLCK/phosphatase system is operative in these cells.

Received October 21, 1996.

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