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Endocrinology Vol. 138, No. 5 1949-1958
Copyright © 1997 by The Endocrine Society

Articles

Mastoparan-Stimulated Prolactin Secretion in Rat Pituitary GH3 Cells Involves Activation of Gq/11 Proteins

Yukiko Yajima, Kazuhiro Uchino, Hisashi Ito and Seiichi Kawashima

Department of Molecular Biology (Y.Y., K.U., S.K.), The Tokyo Metropolitan Institute of Medical Science (Rinsho-ken), 3-18-22, Honkomagome Bunkyo-ku, Tokyo 113, Japan; Department of Chemistry (K.U., H.I.), Aoyama-Gakuin University, 6-16-1, Chitosedai, Setagaya-ku, Tokyo 157, Japan

Address all correspondence and requests for reprints to: Dr. Yukiko Yajima, Department of Molecular Biology, The Tokyo Metropolitan Institute of Medical Science (Rinsho-ken), 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113, Japan. E-mail: yajima{at}rinshoken.or.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mastoparan has been reported to induce a wide variety of cellular actions by activating GTP-binding proteins (G proteins) in various cells. Here, we demonstrate that mastoparan is able to stimulate the secretion of PRL from rat anterior pituitary tumor GH3 cells in dose- and time-dependent manners. Mastoparan had no effect on the accumulation of intracellular cAMP; however, it induced a rapid increase in the intracellular Ca2+ concentration in GH3 cells. Extracellular Ca2+ was required for mastoparan-induced PRL secretion, which was inhibited by nifedipine, an L-type Ca2+ channel blocker. Incubation of mastoparan with myo-[3H]inositol-labeled GH3 cells also resulted in the increased formation of inositol phosphates (InsPs) compared with control cells. Neomycin sulfate and U73122, both phospholipase C inhibitors, suppressed mastoparan-induced PRL secretion. Guanosine 5'-[ß-thio]diphosphate (GDPßS) encapsulated in GH3 cells by reversible electropermeabilization suppressed the response to mastoparan. However, pretreatment with pertussis toxin had no effect on the stimulation of PRL secretion by mastoparan, and both Mas7 (a highly active analogue of mastoparan) and Mas17 (an inactive analogue) enhanced the secretion of PRL to a similar level to that of mastoparan-induced GH3 cells. In contrast, the substance P-related peptide GPant-2A, a Gq antagonist, inhibited mastoparan-induced PRL release, whereas GPant-2, a Gi/o antagonist, did not in electropermeabilized GH3 cells. Moreover, a specific Gq/11 antibody against the carboxyl terminus of the Gq/11 {alpha}-subunit blocked the stimulatory effect of mastoparan on secretion and mastoparan-stimulated InsPs production in digitonin-permeabilized GH3 cells. These results indicate that mastoparan induces the Ca2+-regulated secretion of PRL from GH3 cells by activating Gq/11 and the phospholipase C pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXOCYTOTIC secretion, like other cellular activities, can be modulated by GTP binding proteins (G proteins). Typically, G proteins are involved in transmembranous signaling and mediation between activated transmembranous receptors and effectors that generate intracellular signals (1). There is also growing evidence that G proteins have the ability to regulate secretion independently of all known intracellular signals. Results obtained with permeabilized secretory cells show that G proteins are involved in exocytosis at the distal stages of signal transduction in several cell types including neutrophils (2), mast cells (3), platelet (4), AtT-20 cells (5), RINm5F cells (6), and parathyroid cells (7). In permeable rat pituitary GH3 cells, the possible role of G proteins in the exocytosis of PRL is not yet clear, although ATP-dependency and Ca2+-sensitivity are well recognized in relation to PRL release (8). Ronning and Martin (8) concluded that a poorly nonhydrolyzable GTP analogue, GTP{gamma}S, is a weak stimulator of PRL release, whereas the addition of GTP{gamma}S increases the Ca2+ sensitivity of PRL secretion in electropermeabilized GH3 cells. Recently it has been shown that a blockage of the translation of Rab3b, a low molecular weight G protein, by antisense oligonucleotides leads to the inhibition of exocytosis in PRL-secreting anterior pituitary cells (9). In addition to small G proteins, trimeric-G proteins are known to be involved in various parts of the secretary machinery including formation of secretory vesicles from the Golgi complex, endosome fusion, and regulation of secretory granules (10, 11, 12). Because both heterotrimeric and monomeric G proteins are associated with the membranes of secretory granules in pituitary cells (9, 13, 14), the type of G proteins involved in the exocytotic machinery in pituitary cells remains an open question.

Mastoparan, an amphiphilic tetradecapeptide (Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu-NH2) from wasp venom, forms an {alpha}-helical conformation in phospholipid membranes (15) and stimulates secretion from a number of cell types, including mast cells (16), anterior pituitary cells (17), pancreatic islets of Langerhans (18), and adrenal chromaffin cells (19). Mastoparan is known to activate the GTPase of the heterotrimeric G proteins, Gi and Go, specifically in cell-free systems (20) and interact with the carboxyl terminal domain of the {alpha}-subunits of these G proteins (21). Therefore, mastoparan is believed to interact with Gi/o, pertussis toxin-sensitive G proteins, and affect cellular functions. In addition, mastoparan affects G protein-linked intracellular effector systems such as phospholipase A2 (22), phospholipase C (23), adenylate cyclase (24), and [Ca2+]i (25). Mastoparan also could influence secretory processes by effects which may not be directly mediated by heterotrimeric G proteins, including interactions with low molecular weight G proteins (26) and membrane fusogenic effects (11). Therefore, mastoparan is often used as a tool in the study of the exocytosis mechanism in endocrine cells such as insulin-secreting ß cells (27) and chromaffin cells (12, 19).

For the present report, we studied the effect of mastoparan on the secretion of PRL from rat anterior pituitary tumor GH3 cells and examined whether there is a functional link between the stimulatory effect of mastoparan on PRL secretion and the activation of G proteins. The effect of mastoparan on PRL secretion from pituitary cells has been previously examined by Mau et al. (28). They reported that mastoparan stimulates the secretion of PRL from rat anterior, lactotroph-enriched pituitary cells in a pertussis toxin-insensitive manner that leads to the activation of the phospholipase C/protein kinase C pathway. However, they did not identify the G protein through which mastoparan stimulates PRL secretion from pituitary cells. In the present experiments, we found that mastoparan stimulates PRL secretion from GH3 cells in a pertussis toxin-insensitive manner, but that this effect is inhibited by GDPßS and a Gq antagonist, GPant-2A. We also show that a specific Gq/11 antibody inhibits mastoparan-induced PRL secretion and the formation of mastoparan-stimulated inositol phosphates from digitonin-permeabilized GH3 cells. These results indicate that mastoparan can interact with Gq/11, pertussis toxin-insensitive G proteins, and that Gq/11 may be involved in the exocytotic pathway in GH3 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Drugs and chemicals were obtained from the following sources: Mastoparan, Mas7, Mas17, TRH, VIP, and SRIF, Peptide Institute Co. (Osaka, Japan); U73122, U73343, GPant-2, GPant-2A, isotetrandrine, Biomol. Inc. (Plymouth, PA); Quin-2/AM, DOJINDO Lab. (Kumamoto, Japan); pertussis toxin, digitonin, Wako Pure Chemicals (Osaka, Japan); Neomycin, Sigma Chemical Co. (St. Louis, MO); Gq/11 antibody (catalog no. sc-392), carboxyl terminal Gq/11 {alpha} peptide (catalog no. sc-392P), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The cAMP assay kit for RIA was obtained from Yamasa, Co. (Noda, Japan) and myo-[3H]inositol (80 Ci/mmol) came from New England Nuclear (Boston, MA).

Cell culture
GH3 cells were obtained from American Type Culture Collection (Rockville, MD). The cells were maintained in monolayer culture in Ham’s F-10 medium supplemented with 15% horse serum and 2.5% FBS. For use in experiments, cells from a single donor culture were grown in 35 x 10 mm sterile plastic dishes (Falcon Plastic, CA) on which cells were plated at an initial density of 1.5 x 105 cells/dish; cells were used after 5 or 6 days of subcultivation (60–80% confluence).

PRL secretion from intact cells
Secretion experiments were conducted after 5 days of growth. Two types of medium were used during secretion studies, serum-free Ham’s F-10 medium supplemented with 5 mg/ml lactalbumin, 20 mM HEPES (pH 7.5), or basal salt solution with glucose (BSSG; 10 mM HEPES, pH 7.5, 5 mM KCl, 140 mM NaCl, 1 mM MgCl2, 5 mM glucose, 14 mM NaHCO3, 0.1% BSA). Ca2+ (2 mM)-BSSG was formulated with 2 mM CaCl2 addition. Ca2+-free BSSG was formulated without CaCl2 addition and was found to contain less than 2.5 µM free Ca2+. When supplemented with 100 µM EGTA, this medium contains less than 0.1 µM free Ca2+ (29). Before a secretion experiment, all dishes were preincubated for 30 min in serum-free Ham’s F-10 medium. Then, the medium was removed and 1.0 ml of fresh medium containing test reagents was added, and the cells were incubated at 37 C for 30 min in a 5% CO2 atmosphere. The amount of PRL released into the medium was measured by a specific RIA using a double-antibody technique. Rat PRL assay kits were kindly supplied by Dr. A. F. Parlow, NIADDK, National Hormone and Pituitary Program. The protein contents of the cell residues were determined by a protein assay kit (Bio-Rad Laboratory, Co.) using BSA as a standard.

PRL secretion from cells permeabilized by electric field discharge
Cells were harvested with 0.02% EDTA and then washed twice in buffered saline solution (BSS; 135 mM NaCl, 4.5 mM KCl, 0.5 mM MgCl2, 5.6 mM glucose, 10 mM HEPES, pH 7.5). The last wash and the subsequent steps were carried out at 4 C. The washed cells were resuspended at 1.2 x 107 cells/ml in K+-glutamate buffer (20 mM HEPES, pH 7.5, 5 mM glucose, 2 mM EGTA, 0.1% BSA, 120 mM K+-glutamate, 20 mM NaCl) supplemented with test reagents. Cells were permeabilized by subjecting them to six successive discharges of an electric field (0.8 KV/0.5 ml in 0.4-cm cuvette, 3 µF) using a Gene Pulser (Bio-Rad). Permeabilization was 85–90% as assessed by trypan blue staining. Cells were allowed to reseal for 30 min at 37 C and, after centrifugation, were finally resuspended in K+-glutamate release buffer (20 mM HEPES pH 7.5, 5 mM glucose, 1 mM CaCl2, 0.1% BSA, 120 mM K+-glutamate, 20 mM NaCl, 3 mM MgSO4, 2 mM ATP) and the effects of mastoparan on PRL secretion were tested.

PRL secretion from digitonin-permeabilized cells
Digitonin-permeabilized cells were prepared essentially as described by Ohara-Imaizumi et al. (30). Cells were incubated with medium A containing 140 mM K+-glutamate, 20 mM piperazine N,N'-bis(2-ethanesulfonic acid) (PIPES), 5 mM glucose, 5 mM EGTA, and 0.01 mM digitonin with the pH adjusted to 6.8 with NaOH, with or without Gq/11 antibody (30 µg/ml) for 10 min at 37 C. This permeabilization medium was then replaced with medium B (140 mM K+-glutamate, 20 mM PIPES, 5 mM glucose, 5 mM MgSO4, 5 mM ATP, 1 µM CaCl2, 0.1% BSA) with or without Gq/11 antibody and the suspensions were incubated for an additional 10 min. Subsequently, cells were resuspended in fresh medium B and the effects of mastoparan on PRL secretion were tested.

Measurement of intracellular Ca2+ concentration
We used a similar method for the measurement of [Ca2+]i employing intracellularly trapped quin-2 (31). Briefly, cells were harvested with 0.02% EDTA, washed, and suspended in BSS. Ca2+ (2 mM)-BSS was formulated similarly with 2 mM CaCl2 addition. Cells (2 x 107 cells/ml) were loaded with quin-2 by incubating in Ca2+ (2 mM)-BSS containing 15 µM quin 2/AM (tetraacetoxymethyl-ester of quin-2) at 37 C. After 45 min, the cells were centrifuged at 1000 x g for 3 min, washed, and resuspended in fresh Ca2+ (2 mM)-BSS. The fluorescence intensity of the cell suspension was monitored continuously in a Fluorescence Spectrophotometer F-2000 (Hitachi, Ltd., Ibaragi, Japan) at an excitation wavelength of 340 nm and an emission wavelength of 493 nm. Upon completion of the measurement, 0.1% Triton X-100 was added to measure the maximum fluorescence (Fmax), excess alkaline (pH > 8.5) EGTA was added to measure the minimum fluorescence (Fmin), and finally 5 mM CaCl2 was added to double-check the Fmax. [Ca2+]i was calculated from the following equation: [Ca2+]i = Kd x (F-Fmin)/(Fmax-F), using a value of 115 nM for the Kd of quin-2.

Determination of intracellular cAMP
Cells were preincubated with Ham’s F-10 medium supplemented with 5 mg/ml lactalbumin and 0.5 mM isobutylmethylxanthine. After a 15-min preincubation, the medium was removed and 1.0 ml of fresh medium containing test reagents was added. The cells were incubated for 10 min at 37 C in a 5% CO2 atmosphere. Cellular cAMP was determined by a sensitive RIA procedure as described before (32).

Determination of InsP
We used a similar method for the measurement of [3H]inositol phosphates (33). GH3 cells after 5 days of growth in 35-mm plastic plates were labeled with 2–5 µCi myo-[3H]inositol/ml in inositol-free Ham’s F-10 medium supplemented with 0.3% BSA for 48 h. Cells were preincubated in Ca2+ (1 mM)-BSSG in the presence of 10 mM LiCl for 15 min and then incubated with mastoparan for the indicated times. The reaction was stopped by the rapid addition of 10% HClO4 and neutralized by the addition of 1.53 M KOH/75 mM HEPES. The solution was kept on ice for 1 h, transferred from tray to microtube, and centrifuged at 12,000 x g for 10 min at 4 C. The supernatant was applied to a column of Dowex AG 1-X8 (formate form, 100–200 mesh, Muromachi Chemical Co., Tokyo, Japan). The column was then eluted in a stepwise manner successively with (a) 2 x 8 ml of 60 mM ammonium formate, 5 mM sodium tetraborate; (b) 2 x 8 ml of 0.2 M ammonium formate, 0.1 M formic acid; (c) 2 x 6 ml of 0.4 M ammonium formate, 0.1 M formic acid; (d) 3 x 2 ml of 1 M ammonium formate, 0.1 M formic acid. The radioactivities in fractions b–d were determined in Aquasol (New England Nuclear) in a scintillation spectrometer as the 3H contents of inositol 1-mono-, 1,4-bis-, and 1,4,5-trisphosphates, respectively.

Presentation of the data
PRL release experiments and [3H]InsPs production experiments were performed on at least three different cell preparations. In the figures, which are representative of a single typical experiment, data are given as the mean of triplicate determinations on the same cell preparation ± SE. Differences between means were analyzed by Student’s unpaired t test. A probability of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of mastoparan on the secretion of PRL from GH3 cells
As shown in Fig. 1AGo, mastoparan stimulated the secretion of PRL from GH3 cells in a time-dependent manner. The effect of mastoparan (5 µM) was found to be significant (P < 0.05) after 10 min of incubation and caused a maximum increase in PRL secretion to 160% of the basal value after 30 min of incubation. Mastoparan enhanced the PRL secretion from cells in a dose-dependent manner (ED50 = 3.3 ± 0.6 µM) and the maximum effect was attained at 5 µM (Fig. 1BGo). Concentrations of mastoparan higher than 10 µM were toxic to GH3 cells and resulted in cell death by cell lysis as verified by trypan blue uptake. However, the stimulatory effect of mastoparan was completely reversible, and cell viability was well preserved. After stimulation with 5 µM of mastoparan, and subsequent washout, basal secretion rates and response to VIP (vasoactive intestinal polypeptide) were similar to control, prestimulation values (Table 1Go). Furthermore, mastoparan-induced PRL secretion was temperature-dependent and totally inhibited by incubation at 4 C (Table 1Go). On the other hand, as shown in Fig. 1CGo, mastoparan stimulated the secretion of PRL to a level similar to that produced by TRH or VIP, which are the standard agonists for GH3 cells. SRIF (GH-release-inhibiting factor), a potent inhibitor of PRL release in response to VIP and TRH, inhibited the stimulatory effect of mastoparan by 75% (P < 0.01). It has been reported that SRIF binds to specific receptors and inhibits adenylate cyclase through Gi protein (32, 34). As shown in Fig. 1DGo, however, mastoparan did not stimulate intracellular cAMP production.



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  		Figure 1. Characterization of mastoparan (MP) action on GH3 cells. A, Time course for mastoparan action on PRL secretion. GH3 cells (1.5 x 105 cells) were cultured at 37 C in growth medium for 5 days, and the medium was changed to experimental medium with (closed circles) or without (open circles) mastoparan (5 µM). The cells were incubated for various times as indicated at 37 C. The medium was collected for the measurement of PRL concentration as described under Experimental procedures. B, Concentration dependency of mastoparan on PRL secretion. Cells were incubated in experimental medium containing the indicated concentrations of mastoparan for 30 min at 37 C and the released PRL was determined. C, Effects of mastoparan and secretory reagents on PRL secretion. Experimental medium was added to cells with mastoparan (5 µM), VIP (0.1 µM), TRH (0.1 µM), or SRIF (0.1 µM). The cells were incubated for 30 min at 37 C and the released PRL was determined. D, Cells were incubated in medium containing 0.5 mM isobutylmethylxanthine for 30 min at 37 C. The medium was changed then to fresh experimental medium containing mastoparan (5 µM), VIP (0.1 µM), TRH (0.1 µM), or SRIF (0.1 µM). Cells were incubated for 10 min at 37 C, and the reaction was terminated by rapid aspiration of the medium. cAMP content in cells was analyzed as described in Experimental procedures. Values represent the means ± SE of triplicate determinations in one representative experiment. The significant differences (*, P < 0.05; **, P < 0.01) in PRL secretion from the appropriate control are shown.

 

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  		Table 1. Characterization of mastoparan action on GH3 cells

 
Evidence for Ca2+-regulated secretion of mastoparan action
We studied the effect of mastoparan on intracellular Ca2+ concentrations in quin-2 loaded GH3 cells. The addition of mastoparan to quin-2 loaded GH3 cells resulted in a prominent increase in intracellular Ca2+ concentrations within 5–10 sec, after which the Ca2+ concentrations gradually returned to basal levels over the next 80–100 sec (Fig. 2AGo). The intracellular Ca2+ concentration increased from basal levels of 60–160 nM to 220–430 nM upon challenge with mastoparan. The increase in intracellular Ca2+ was dependent on the concentration of mastoparan. The mastoparan (5 µM)-stimulated increase in intracellular Ca2+ was considerably less but still induced in cells in Ca2+-free medium (43 ± 7 nM, n = 4) or in cells pretreated with nifedipine, an L-type calcium channel blocker (48 ± 16 nM, n = 4). Mau et al. (28) also showed that mastoparan is able to increase the intracellular Ca2+ concentration of rat anterior pituitary cells even in the absence of Ca2+ from the extracellular medium (28). These results indicate that mastoparan causes a release of Ca2+ from intracellular stores and an influx of extracellular Ca2+. On the other hand, when extracellular Ca2+ concentrations were low ([Ca2+] < 2.5 µM), a small, statistically insignificant effect on PRL secretion was detected in mastoparan-treated cells (Fig. 2BGo). However, when 2 mM CaCl2 was added to the buffer, mastoparan stimulated the secretion of PRL to 140% of control levels. Moreover, when 2 mM CaCl2 and 2 µM nifedipine were added to the buffer, the effect of mastoparan was inhibited completely (P < 0.01). Basal secretion was not significantly affected by nifedipine treatment.



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  		Figure 2. Relationship between mastoparan (MP) and intra- and extracellular Ca2+ in GH3 cells. A, Effect of mastoparan on intracellular Ca2+ in GH3 cell. Free calcium concentration was measured in quin-2-loaded GH3 cells as described in Experimental procedures. Quin-2-loaded GH3 cells were incubated at 37 C in Ca2+ (2 mM)-BSS and the change in Ca2+ concentration was recorded after the addition of 2.5, 1, or 5 µM mastoparan. Mastoparan was added at the times indicated by the arrows. B, Calcium dependency of mastoparan-stimulated PRL secretion. All release experiments were conducted in BSSG medium. The cells were preincubated with Ca2+-free BSSG supplemented with 100 µM EGTA for 30 min. Subsequently, the cells were incubated without or with mastoparan (5 µM) or TRH (0.1 µM) in Ca2+-free BSSG, 2 mM Ca2+ BSSG, or 2 mM Ca2+ BSSG with 2 µM nifedipine for 30 min at 37 C. The supernatant was collected for the measurement of PRL concentration. Values represent the means ± SE of triplicate determinations in one representative experiment. The significance (P < 0.01) of the difference in PRL secretion caused by mastoparan between cells in 2 mM Ca2+ BSSG and 2 mM Ca2+ BSSG with nifedipine is shown.

 
Role of phospholipase C on mastoparan-stimulated PRL secretion from GH3 cells
Next we tested the formation of [3H]inositol phosphates by mastoparan in myo-[3H]inositol-labeled GH3 cells. Mastoparan (5 µM) induced the accumulation of inositol trisphosphate (InsP3), inositol bisphosphate (InsP2), and inositol monophosphate (InsP1) in a time-dependent manner (Fig. 3Go). InsP3 was the first product that accumulated, reaching a maximum at 30 sec of incubation, whereas the levels of InsP2 and InsP1 increased slowly over the same time course. On the other hand, Neomycin (100 µM) and U73122 (5 µM), both phospholipase C inhibitors (35, 36), suppressed mastoparan-induced PRL release, whereas U73343 (5 µM), a negative control for U73122, did not (Table 2Go). Isotetrandrine (10 µM), a G protein-coupled phospholipase A2 inhibitor (37), did not suppress the effect of mastoparan in GH3 cells (Table 2Go). These results indicate that mastoparan induces PRL secretion by stimulating the activity of phospholipase C, but not phospholipase A2, in GH3 cells.



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  		Figure 3. Time course of mastoparan-stimulated inositol phosphate production in GH3 cells. GH3 cells prelabeled with 2 µCi myo-[3H]inositol/ml in 35-mm dishes were stimulated with 5 µM mastoparan at 37 C for the indicated times in the presence of 10 mM LiCl. Data were corrected for blanks incubated without mastoparan addition and are shown as the mean ± SE of quadruplicate determinations in a representative experiment. *, P < 0.05; **, P < 0.01 (significantly different from 0 sec incubation).

 

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  		Table 2. Effects of neomycin, U73122, U73343, and isotetrandrine on mastoparan stimulation of PRL secretion in GH3 cells

 
Identification of the G proteins responsible for mastoparan-stimulated PRL secretion from GH3 cells
In order to confirm the involvement of G proteins in mastoparan action, we examined the effects of GTP{gamma}S (an active GTP analogue) and GDPßS (an inactive GDP analogue) on the mastoparan-induced secretion of PRL in electropermeabilized and resealed GH3 cells. The introduction of GTP{gamma}S or GDPßS into the cytosolic compartments of GH3 cells by reversible electropermeabilization did not modify basal prolactin secretion (Fig. 4AGo). GTP{gamma}S might not bind to some G proteins, e.g. Gq/11, without the input of an agonist (38). In contrast, GTP{gamma}S somewhat promoted mastoparan action, but GDPßS strongly suppressed PRL secretion in response to mastoparan, with levels decreasing from 175% to 105% (Fig. 4AGo). The ability of GDPßS to attenuate the stimulatory action of mastoparan was found to be dependent on the concentration of GDPßS (Fig. 4BGo). Next, we studied the target G proteins in mastoparan action on GH3 cells. Although pretreatment with pertussis toxin (50 ng/ml) for 24-h inhibited SRIF action on mastoparan-stimulated PRL secretion, it did not affect cell response to mastoparan (5 µM) (Fig. 5AGo). On the other hand, both Mas7 (a mastoparan analogue highly active in stimulating heterotrimeric Gi/o proteins, Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Ala-Leu-Leu-NH2) and Mas17 (an inactive analogue, Ile-Asn-Leu-Lys-Ala-Lys-Ala-Ala-Leu-Ala-Lys-Lys-Leu-Leu–NH2) stimulated the secretion of PRL to levels similar to that of mastoparan (Fig. 5BGo). These results indicate that mastoparan can stimulate the secretion of PRL by activating G proteins other than Gi and Go. In searching for G proteins that interact with mastoparan, we studied the possibility that mastoparan interacts with Gq/11, which is involved in the activation of phospholipase C in GH3 cells (39), using a specific Gq antagonist, GPant-2A (Fig. 6Go). It has been reported that a substance P-related peptide, GPant-2A (Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2), potently inhibits GTP hydrolysis of Gq by activating the M1 muscarinic cholinergic receptor and inhibits the effect of a CCK (cholecystokinin) receptor agonist, CCK-OPE, which is coupled to Gq/11 proteins, in pancreatic acini (40, 41). Encapsulation of 30 µM GPant-2A during reversible electropermeabilization suppressed mastoparan-stimulated hormone secretion from 200 to 125% of control values and also inhibited the effect of TRH, which is reported to bind specific receptors and stimulate phospholipase C through Gq/11 proteins (Fig. 6AGo) (39). However, cells treated with another G protein antagonist, GPant-2 (Pyr-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2), which inhibits GTP hydrolysis by Gi/o (40) and suppresses the effect of mastoparan or GAP-43 on regulated exocytosis by activating Go (12, 42), showed no changes in their responses to mastoparan and TRH compared with control cells (Fig. 6AGo). However, GPant-2 (30 µM) reduced the inhibitory effect of SRIF against the secretory effect of VIP in GH3 cells (from 57.4 ± 5.7% to 94.2 ± 10.8%, P < 0.05 n = 4). This shows the difference in the specificities of GPant-2A and GPant-2. The effect of GPant-2A suppressing mastoparan-stimulated hormone secretion was dose dependent (Fig. 6BGo). Mastoparan has been reported to interact with the carboxyl terminus of the {alpha}-subunit of G proteins (21). Therefore, we used a polyclonal antibody raised against the carboxyl terminus of the Gq/11 {alpha}-subunit in an attempt to antagonize the effect of mastoparan in digitonin-permeabilized GH3 cells. As shown in Fig. 7AGo, the Gq/11 antibody (30 µg/ml) did not significantly modify the basal secretion of PRL in digitonin-permeabilized cells. Mastoparan stimulated PRL secretion by 162% in the digitonin-permeabilized cells, and this stimulation was unaffected by the presence of normal rabbit serum (data not shown). However, the stimulatory effect of mastoparan was suppressed to only 14% in the presence of the Gq/11 antibody. Preincubation of the Gq/11 antibody with the Gq/11{alpha} peptide (6 µg/ml) before its addition to cells abolished the effect of the antibody, indicating that the Gq/11 antibody blocks the effect of mastoparan on PRL secretion by binding specifically to endogenous Gq/11. Mastoparan also stimulated the formation of [3H]inositol phosphates in digitonin-permeabilized GH3 cells prelabeled with myo-[3H]inositol (Fig. 7BGo). In these preparations, the accumulation of InsP2 and InsP3 was observed, due to the fact that the enzyme that dephosphorylates InsP2 to InsP1 is cytosolic and therefore is not recognized in digitonin permeabilized cells (43). The Gq/11 antibody was able to block the mastoparan-induced hydrolysis of [3H]inositol phospholipids in myo-[3H]inositol-labeled GH3 cells. The stimulatory effect of mastoparan on the formation of [3H]inositol phosphates was reduced to only 27% in the presence of the Gq/11 antibody, whereas the Gq/11 peptide abolished the effect of the Gq/11 antibody. These results clearly indicate that the target protein of mastoparan in GH3 cells is a protein of the Gq/11 family and that mastoparan induces the Ca2+-regulated secretion of PRL by activating Gq/11 and phospholipase C.



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  		Figure 4. Inhibitory effect of GDPßS on mastoparan (MP)-stimulated PRL release from electropermeabilized GH3 cells. A, GH3 cells were permeabilized by electropermeabilization as described in Experimental procedures. Cells containing encapsulated GTP{gamma}S (1 mM) or GDPßS (1 mM) were incubated with or without mastoparan (5 µM) for 30 min at 37 C. The supernatants were collected for the measurement of PRL concentration. B, Dose dependency of GDPßS on the stimulatory effect of mastoparan in electropermeabilized GH3 cells. Values represent the means ± SE of triplicate determinations in one representative experiment. The significance (P < 0.01) of the difference in PRL secretion caused by mastoparan with and without GDPßS treatment is shown.

 


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  		Figure 5. Effect of pertussis toxin (PT) on mastoparan (MP)-stimulated PRL secretion and mastoparan analogues on PRL secretion from GH3 cells. A, Effect of PT on the stimulation of PRL secretion by mastoparan. GH3 cells were treated with (closed bars) or without (open bars) PT (50 ng/ml) for 24 h. New experimental medium was added to the cells with or without mastoparan (5 µM) or SRIF (0.1 µM). The cells were incubated for 30 min and the PRL concentration in the supernatant was analyzed as described under Experimental procedures. The significance (P < 0.01) of the difference in PRL secretion caused by mastoparan plus SRIF (0.1 µM) with and without PT treatment is shown. B, Effect of mastoparan analogues on PRL secretion from GH3 cells. GH3 cells were incubated with mastoparan (5 µM), Mas7 (5 µM), or Mas17 (5 µM) for 30 min. Values represent the means ± SE of triplicate determinations in one representative experiment.

 


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  		Figure 6. Effects of G protein antagonists on mastoparan (MP) stimulation of PRL secretion from electropermeabilized GH3 cells. A, GPant-2 (30 µM) or GPant-2A (30 µM) was encapsulated inside electrically permeabilized GH3 cells and the cells were incubated with or without mastoparan (5 µM) or TRH (0.1 µM) for 30 min at 37 C. The supernatants were collected for the measurement of PRL concentration. *, The significance (P < 0.01) of the differences in PRL secretion between cells with GPant-2A treatment and those without is shown. B, The GPant-2A dose-dependency on the stimulatory effect of mastoparan in electropermeabilized GH3 cells. Values represent the means ± SE of triplicate determinations in one representative experiment.

 


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  		Figure 7. Effect of Gq/11 antibody on the mastoparan (MP) stimulation of PRL secretion from digitonin-permeabilized GH3 cells and MP-induced formation of [3H]InsPs in myo-[3H]inositol-labeled GH3 cells. A, Digitonin-permeabilized cells were exposed to Gq/11 antibody (30 µg/ml) as described in Experimental procedures. Subsequently, the digitonin-permeabilized cells were incubated with or without mastoparan (5 µM) for 30 min at 37 C. The supernatant was collected for the measurement of PRL concentration. Preincubation of the Gq/11 antibody (30 µg/ml) with the carboxy terminal Gq/11 {alpha} peptide (6 µg/ml) was performed for 2 h at 4 C before the addition of the antibody to cells as described under Experimental procedures. B, myo-[3H]inositol-labeled GH3 cells were permeabilized by digitonin and exposed to Gq/11 antibody (30 µg/ml) as described in A. Mastoparan was then added for 30 min at a concentration of 5 µM in the presence of 10 mM LiCl. The radioactivity of the total InsPs fraction (containing InsP2 and InsP3) is shown. Values represent the means ± SE of triplicate determinations. *, The significance (P < 0.01) of the difference in PRL secretion or [3H]InsPs formation caused by mastoparan between cells with Gq/11 antibody treatment and those without is shown.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present data show that mastoparan stimulates the secretion of PRL from the rat pituitary clonal cell line GH3. This process is dose dependent and time dependent and requires the presence of extracellular Ca2+. In a concentration range of 0.1–10 µM, mastoparan has no effect on cell viability. In GH3 cells, the effect of the peptide, at least at concentrations up to 10 µM, is caused by stimulated exocytosis, not nonspecific cellular destruction. We list the following as the basis for this conclusion. 1) The number and gross microscopic appearance of cells after stimulation with various concentrations of mastoparan up to 10 µM showed no significant changes. Higher concentrations of the peptide (>20 µM) sometimes decreased the number of attached cells after static incubation. 2) Basal PRL secretion and prolactin release response to VIP, after exposure to mastoparan and subsequent removal by washing, was the same as for the control cells. 3) Lowering the ambient temperature to 4 C greatly suppressed 5 µM mastoparan-induced PRL release. 4) Mastoparan-induced PRL secretion was inhibited by GDPßS, phospholipase C inhibitor, Gq antagonist, and a specific Gq/11 antibody, none of which would be expected to inhibit a nonspecific cytolytic process, suggesting that these concentrations of mastoparan do not affect GH3 cell integrity. Mastoparan also stimulates the secretion of GH in GH3 cells (data not shown). The effect of mastoparan is comparable to that of TRH and VIP, which activate different initial events to stimulate hormone secretion from GH3 cells. SRIF also suppresses the stimulation of PRL secretion by mastoparan in GH3 cells. The present data showing that mastoparan has no effect on intracellular cAMP concentrations but increases the intracellular Ca2+ concentration suggest that SRIF inhibits the effect of mastoparan by attenuating the intracellular Ca2+ concentration. Therefore, the action of mastoparan appears to be similar to that of TRH in that it increases the intracellular Ca2+ concentration and subsequently stimulates the secretion of PRL in GH3 cells. However, the present experiments show a difference between the effects of mastoparan and TRH in that nifedipine, an L-type-Ca2+ channel blocker, completely inhibits the effect of mastoparan but suppresses moderately the effect of TRH on PRL secretion. These results indicate that the stimulation of PRL secretion by mastoparan is due to an increase in intracellular Ca2+ caused by the influx of extracellular Ca2+ through the L-type Ca2+ channel rather than to a release from intracellular Ca2+ stores for hormone secretary mechanisms as in the case of TRH. Furthermore, the present results indicate that Ca2+ channels and phospholipase C, which produces inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, are indeed active in the mastoparan-stimulated secretory process in intact GH3 cells. Evidence from a variety of cell types indicates that the enhanced entry of extracellular Ca2+ is associated in some way with the receptor-activated generation of InsP3 (44). However, there are suggestions that InsP3 may have direct effect on calcium-permeable channels, not voltage-dependent channels (45). On the other hand, it has been reported that diacylglycerol induces the influx of extracellular calcium in GH4C1 cells, possibly by activating a voltage-dependent Ca2+ channel (46). Therefore, it seems that mastoparan activates voltage-dependent calcium channels by phospholipase C-generated diacylglycerol in GH3 cells. However, this conclusion needs confirmation by direct electrophysiological characterization of the Ca2+ currents induced by mastoparan. On the other hand, in digitonin-permeabilized GH3 cells, neither EGTA nor nifedipine blocked the mastoparan-induced PRL secretion (unpublished data). Therefore, it is feasible to speculate that mastoparan activates Gq/11 proteins that stimulate exocytosis beyond the elevation of [Ca2+]i in permeabilized GH3 cells.

In this study, we have presented data that strongly suggest that mastoparan interacts with G proteins, especially Gq/11 proteins, in the secretory process of PRL in GH3 cells, because GDPßS in the present experiments consistently inhibited mastoparan-induced PRL secretion in electropermeabilized GH3 cells. Furthermore, the effect of mastoparan on PRL release in GH3 cells was inhibited dose dependently by GPant-2A, an antagonist of Gq and by a Gq/11-specific antibody. Mastoparan has been shown to increase the GTPase activity of heterotrimeric G proteins, especially Gi and Go (20). Also, we found that mastoparan interacted with Gi/o in GH3 cells in vitro because the effect of mastoparan on GTPase activity of GH3 cell membranes was suppressed in the membranes from cells treated with pertussis toxin (data not shown). However, there are some conflicting reports to suggest that all of mastoparan action is not due to the stimulation of Gi or Go activity as a simple mimic of G protein-linked agonist-liganded receptor. For example, the action of mastoparan on the stimulatory secretion of PRL from cultured rat anterior pituitary cells (28), the stimulation of vascular reactivity in hypertensive rats (47) and PI turnover in rat hepatocytes (48), and the inhibition of PI turnover in human astrocytoma cells (49) are not attenuated by pretreatment with pertussis toxin. In the present experiments, we showed that mastoparan causes PRL secretion without any interaction with Gi/o or Gs because neither pertussis toxin nor GPant-2 suppressed mastoparan action, and mastoparan did not stimulate the accumulation of intracellular cAMP. Moreover, we found that both Mas7 and Mas17 enhance the secretion of PRL and that no significant difference could be detected between the activity of Mas7 and Mas17. Higashijima et al. have developed these active and inactive analogues depending on the formation of amphipathic {alpha}-helical conformations in the presence of phospholipid membranes (20). However, they examined these analogues only for Gi/o, Gs and Gt, so it is not clear whether these analogues can be applied to other G proteins in which the existence of at least 20 {alpha}-subunits has been revealed. On the other hand, Voss et al. (50) have reported that an amphipathic {alpha}-helix does not represent the main structural determinant for the receptor-G protein interaction site. Nevertheless, the possibility can not be excluded that mastoparan may activate Gq/11 family members indirectly through NDP kinase, which catalyzes the conversion of GDP to GTP, because mastoparan, Mas7, and Mas17 each activate NDP kinase in normal rat islets (51) like they stimulate PRL secretion in GH3 cells (Fig. 5Go). At present, it is not known why mastoparan does not interact with Gi/o in intact GH3 cells. It seems possible that activation of multiple G proteins in the cells by mastoparan results in a variety of actions determined by the cell, its G protein composition and its intracellular environment. For example, there are some differences between pituitary cells and others in such as the response to rab3AL (33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48), a synthetic peptide of the rab3a effector domain, which enhances regulated exocytosis in permeabilized pancreatic acini (52) and chromaffin cells (53), whereas it inhibits PRL secretion in pituitary cells (54). Clearly, further studies to evaluate the mechanism of mastoparan-G protein interaction in GH3 cells are required.

Mastoparan induces exocytosis in a receptor-independent manner by interacting directly with a variety of G proteins because the G proteins involved in exocytosis vary greatly with cell type, such as Gi3 in the plasma membrane of rat peritoneal mast cells (55), Go in secretory granules of bovine chromaffin cells (12), and Gi in the insulin secretory granules of ß-TC3 cells (27). The present results show that mastoparan stimulates PRL secretion in intact and digitonin-permeabilized cells, and that Gq/11 plays an essential role in mastoparan-mediated PRL secretion, presumably by stimulating phospholipase C in GH3 cells. Wilson et al. (13) have described the cell periphery as the predominant site for the localization of Gs, Gi, and Gq in all glandular cell types of the pituitary. In addition, Gi3 and Gq have been detected in the Golgi region and a small amount of Gs is observed on the secretory granule membrane. It seems that the plasma membrane-bound form of Gq/11 facilitates regulated exocytosis in GH3 cells because mastoparan cannot reach proteins associated with intracellular compartments like Golgi region in intact cells (12). This is the first report that mastoparan could interact with Gq/11, rather than with pertussis toxin-sensitive G proteins of the Gi/o family, and that Gq/11 is involved in the secretory processes in GH3 cells.


   Acknowledgments
 
We thank Dr. M. Ui for critical discussion and Dr. T. Saido and Ms. W. Harigaya for valuable advice about electropermiabilization methods.

Received November 4, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gilman AG 1987 G proteins: transducers of receptor-generated signals. Annu Rev Biochem 56:615–649[CrossRef][Medline]
  2. Barrowman MM, Cockcroft S, Gomperts BD 1986 Two roles for guanine nucleotides in the stimulus-secretion sequence of neutrophils. Nature 319:504–507[CrossRef][Medline]
  3. Howell TW, Cockcroft S, Gomperts BD 1987 Essential synergy between Ca2+ and guanine nucleotides in exocytotic secretion from permeabilized rat mast cells. J Cell Biol 105:191–197[Abstract/Free Full Text]
  4. Haslam RJ, Davidson MM 1984 Guanine nucleotides decrease the free [Ca2+] required for secretion of serotonin from permeabilized blood platelets. Evidence of a role for a GTP binding protein in platelet activation. FEBS Lett 174:90–95[CrossRef][Medline]
  5. Luini A, De Matteis MA 1990 Evidence that receptor-linked G protein inhibits exocytosis by a post-second-messenger mechanism in AtT-20 cells. J Neurochem 54:30–38[Medline]
  6. Vallar L, Biden TJ, Wollheim CB 1987 Guanine nucleotides induce Ca2+-independent insulin secretion from permeabilized RINm5F cells. J Biol Chem 262:5049–5056[Abstract/Free Full Text]
  7. Oetting M, LeBoff M, Swiston L, Preston J, Brown E 1986 Guanine nucleotides are potent secretagogues in permeabilized parathyroid cells. FEBS Lett 208:99–104[CrossRef][Medline]
  8. Ronning SA, Martin TFJ 1986 Characterization of phorbol ester- and diacylglycerol-stimulated secretion in permeable GH3 pituitary cells. Interaction with Ca2+. J Biol Chem 261:7840–7845[Abstract/Free Full Text]
  9. Lledo P-M, Vernier P, Vincent J-D, Mason WT, Zorec R 1993 Inhibition of Rab3B expression attenuates Ca2+-dependent exocytosis in rat anterior pituitary cells. Nature 364:540–544[CrossRef][Medline]
  10. Donaldson JG, Kahn RA, Lippincott-Schwartz J, Klausner RD 1991 Binding of ARF and beta-COP to Golgi membranes: possible regulation by a trimeric G protein. Science 254:1197–1199[Abstract/Free Full Text]
  11. Colombo MI, Mayorga LS, Casey PJ, Stahl PD 1992 Evidence of a role for heterotrimeric GTP-binding proteins in endosome fusion. Science 255:1695–1697[Abstract/Free Full Text]
  12. Vitale N, Mukai H, Rouot B, Thierse D, Aunis D, Bader MF 1993 Exocytosis in chromaffin cells: possible involvement of the heterotrimeric GTP-binding protein Go. J Biol Chem 268:14715–14723[Abstract/Free Full Text]
  13. Wilson BS, Komuro M, Farquhar MG 1994 Cellular variations in heterotrimeric G protein localization and expression in rat pituitary. Endocrinology 134:233–244[Abstract]
  14. Muller L, Picart R, Barret A, Bockaert J, Homburger V, Tougard C 1994 Identification of multiple subunits of heterotrimeric G proteins on the membrane of secretory granules in rat prolactin anterior pituitary cells. Mol Cell Neurosci 5:556–566[CrossRef][Medline]
  15. Higashijima T, Wakamatsu K, Takemitsu M, Fujino M, Nakajima T, Miyazawa T 1983 Conformational change of mastoparan from wasp venom on binding with phospholipid membrane. FEBS Lett 152:227–230[CrossRef][Medline]
  16. Hirai Y, Yasuhara T, Yoshida H, Nakajima T, Fujino M, Kitada C 1979 A new mast cell degranulating peptide "mastoparan" in the venom of Vespula lewisii. Chem Pharm Bull (Tokyo) 27:1942–1944[Medline]
  17. Kurihara H, Kitajima K, Senda T, Fujita H, Nakajima T 1986 Multigranular exocytosis induced by phospholipase A2-activators, melittin and mastoparan, in rat anterior pituitary cells. Cell Tissue Res 243:311–316[Medline]
  18. Yokokawa N, Komatsu M, Takeda T, Aizawa T, Yamada T 1989 Mastoparan, a wasp venom, stimulates insulin release by pancreatic islets through pertussis toxin sensitive GTP-binding protein. Biochem Biophys Res Commun 158:712–716[CrossRef][Medline]
  19. Vitale N, Thierse D, Aunis D, Bader MF 1994 Exocytosis in chromaffin cells: evidence for a MgATP-independent step that requires a pertussis toxin-sensitive GTP-binding protein. Biochem J 300:217–227
  20. Higashijima T, Burnier J, Ross EM 1990 Regulation of Gi and Go by mastoparan, related amphiphilic peptides, and hydrophobic amines: mechanism and structural determinant of activity. J Biol Chem 265:14176–14186[Abstract/Free Full Text]
  21. Weingarten R, Ransnas L, Mueller H, Sklar LA, Bokoch GM 1990 Mastoparan interacts with the carboxyl terminus of the {alpha} subunit of Gi. J Biol Chem 265:11044–11049[Abstract/Free Full Text]
  22. Argiolas A, Pisano JJ 1983 Facilitation of phospholipase A2 activity by mastoparan, a new class of mast cell degranulating peptides from wasp venom. J Biol Chem 258:13697–13702[Abstract/Free Full Text]
  23. Wallace MA, Carter HR 1989 Effects of the wasp venom peptide, mastoparan, on a phosphoinositide-specific phospholipase C purified from rabbit brain membranes. Biochim Biophys Acta 1006:311–316[Medline]
  24. Wheeler-Jones CPC, Saermark T, Kakkar VV, Authi KS 1992 Mastoparan promotes exocytosis and increases intracellular cyclic AMP in human platelets: evidence for the existence of a Ge-like mechanism of secretion. Biochem J 281:465–472
  25. Komatsu M, Aizawa T, Yokokawa N, Sato Y, Okada N, Takasu N, Yamada T 1992 Mastoparan-induced hormone release from rat pancreatic islets. Endocrinol 130:221–228[Abstract]
  26. Koch G, Haberman B, Mohr C, Just I, Aktories K 1991 Interaction of mastoparan with the low molecular mass GTP-binding proteins rho/rac. FEBS Lett 291:336–340[CrossRef][Medline]
  27. Konrad RJ, Young RA, Record RD, Smith RM, Butkerait P, Manning D, Jarett L, Wolf BA 1995 The heterotrimeric G-protein Gi is localized to the insulin secretory granules of ß-cells and is involved in insulin exocytosis. J Biol Chem 270:12869–12876[Abstract/Free Full Text]
  28. Mau SE, Witt MR, Vilhardt H 1994 Mastoparan, a wasp venom peptide, stimulates release of prolactin from cultured rat anterior pituitary cells. J Endocrinol 142:9–18[Abstract]
  29. Martin TFJ, Kowalchyk JA 1984 Evidence for the role of calcium and diacylglycerol as dual second messengers in thyrotropin-releasing hormone action: involvement of Ca2+. Endocrinology 115:1527–1536[Abstract]
  30. Ohara-Imaizumi M, Kameyama K, Kawae N, Takeda K, Muramatsu S, Kumakura K 1992 Regulatory role of the GTP-binding protein, G(o), in the mechanism of exocytosis in adrenal chromaffin cells. J Neurochem 58:2275–2284[CrossRef][Medline]
  31. Gershengorn MC, Thaw C 1983 Calcium influx is not required for TRH to elevate free cytoplasmic calcium in GH3 cells. Endocrinology 113:1522–1524[Abstract]
  32. Yajima Y, Akita Y, Saito T 1986 Pertussis toxin blocks the inhibitory effects of somatostatin on cAMP-dependent vasoactive intestinal peptide and cAMP-independent thyrotropin releasing hormone-stimulated prolactin secretion of GH3 cells. J Biol Chem 261:2684–2689[Abstract/Free Full Text]
  33. Murayama T, Ui M 1987 Phosphatidic acid may stimulate membrane receptors mediating adenylate cyclase inhibition and phospholipid breakdown in 3T3 fibroblasts. J Biol Chem 262:5522–5529[Abstract/Free Full Text]
  34. Koch BD, Schonbrunn A 1984 The somatostatin receptor is directly coupled to adenylate cyclase in GH4C1 pituitary cell membranes. Endocrinology 114:1784–1790[Abstract]
  35. Vergara J, Tsien RY, Delay M 1985 Inositol 1,4,5-trisphosphate: a possible chemical link in excitation-contraction coupling in muscle. Proc Natl Acad Sci USA 82:6352–6356[Abstract/Free Full Text]
  36. Bleasdale JE, Thakur NR, Gremban RS, Bundy GL, Fitzpatrick FA, Smith RJ, Bunting S 1990 Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils. J Pharmcol Exp Ther 255:756–768[Abstract/Free Full Text]
  37. Hashizume T, Yamaguchi H, Sato T, Fujii T 1991 Suppressive effect of biscoclaurine alkaloids on agonist-induced activation of phospholipase A2 in rabbit platelets. Biochem Pharmacol 4:419–423
  38. Helper JR, Kozasa T, Smrcka AV, Simon MI, Rhee SG, Sternweis PC, Gilman AG 1993 Purification from Sf9 cells and characterization of recombinant Gq{alpha} and G11{alpha}. Activation of purified phospholipase C isozymes by G{alpha} subunits. J Biol Chem 268:14367–14375[Abstract/Free Full Text]
  39. Aragay AM, Katz A, Simon MI 1992 The G{alpha}q and G{alpha}11 proteins couple the thyrotropin-releasing hormone receptor to phospholipase C in GH3 rat pituitary cells. J Biol Chem 267:24983–24988[Abstract/Free Full Text]
  40. Mukai H, Munekata E, Higashijima T 1992 G protein antagonists: a novel hydrophobic peptide competes with receptor for G protein binding. J Biol Chem 267:16237–16243[Abstract/Free Full Text]
  41. Tsunoda Y, Owyang C 1995 The regulatory site of functional GTP binding protein coupled to the high affinity cholecystokinin receptor and phospholipase A2 pathway is on the G ß subunit of Gq protein in pancreatic acini. Biochem Biophys Res Commun 211:648–655[CrossRef][Medline]
  42. Vitale N, Deloulme J-C, Thierse D, Aunis D, Bader MF 1994 GAP-43 controls the availability of secretory chromaffin granules for regulated exocytosis by stimulating a granule-associated Go. J Biol Chem 269:30293–30298[Abstract/Free Full Text]
  43. Storey DJ, Shears SB, Kirk CJ, Michell RH 1984 Stepwise enzymatic dephosphorylation of inositol 1,4,5-trisphosphate to inositol in liver. Nature 312:374–376[CrossRef][Medline]
  44. Berridge M 1993 Inositol trisphosphate and calcium signalling. Nature 361:315–325[CrossRef][Medline]
  45. Mochizuki-Oda N, Nakajima Y, Nakanishi S, Ito S 1994 Characterization of the substance P receptor-mediated calcium influx in cDNA transfected chinese hamster ovary cells. A possible role of inositol 1,4,5-trisphosphate in calcium influx. J Biol Chem 269:9651–9658[Abstract/Free Full Text]
  46. Albert PR, Wolfson G, Tashjian AH Jr 1987 Diacylglycerol increases cytosolic free Ca2+ concentration in rat pituitary cells. Relationship to thyrotropin-releasing hormone action. J Biol Chem 262:6577–6581[Abstract/Free Full Text]
  47. Kanagy NL, Webb RC 1994 Enhanced vascular reactivity to mastoparan, a G protein activator, in genetically hypertensive rats. Hypertension 23:946–950[Abstract/Free Full Text]
  48. Tohkin M, Yagami T, Matsubara T 1990 Mastoparan, a peptide toxin from wasp venom, stimulates glycogenolysis mediated by an increase of the cytosolic free Ca2+ concentration but not by an increase of cAMP in rat hepatocytes. FEBS Lett 260:179–182[CrossRef][Medline]
  49. Nakahata N, Abe MT, Matsuoka I, Nakanishi H 1990 Mastoparan inhibits phosphoinositide hydrolysis via pertussis toxin-intensive G-protein in human astrocytoma cells. FEBS Lett 260:91–94[CrossRef][Medline]
  50. Voss T, Wallner E, Czernilofsky AP, Freissmuth M 1993 Amphipathic {alpha}-helical structure does not predict the ability of receptor-derived synthetic peptides to interact with guanine nucleotide-binding regulatory proteins. J Biol Chem 268:4637–4642[Abstract/Free Full Text]
  51. Kowluru A, Seavey SE, Rabaglia ME, Metz SA 1995 Non-specific stimulatory effects of mastoparan on pancreatic islet nucleoside diphosphokinase activity: dissociation from insulin secretion. Biochem Pharmacol 49:263–266
  52. Padfield PJ, Balch WE, Jamieson JD 1992 A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini. Proc Natl Acad Sci USA 89:1656–1660[Abstract/Free Full Text]
  53. Senyshyn J, Balch WE, Holz RW 1992 Synthetic peptides of the effector-binding domain of rab enhance secretion from digitonin-permeabilized chromaffin cells. FEBS Lett 309:41–46[CrossRef][Medline]
  54. Davidson JS, Eales A, Roeske RW, Millar RP 1993 Inhibition of pituitary hormone exocytosis by a synthetic peptide related to the rab effector domain. FEBS Lett 326:219–221[CrossRef][Medline]
  55. Aridor M, Rajmilevich G, Beaven MA, Sagi-Eisenberg R 1993 Activation of exocytosis by the heterotrimeric G proteins Gi3. Science 262:1569–1572[Abstract/Free Full Text]



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