Mastoparan Stimulates Exocytosis at a Ca2+-independent Late Site

THE JOURNAL 0 1993 by The American Society for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc

Vol. 268, No. 31, Issue of November 5, pp. 23297-23306.1993 Printed in U.S.A.

Mastoparan Stimulates Exocytosis at a Ca2+-independent Late Site in Stimulus-Secretion Coupling STUDIES WITH THE RINm5F &CELL LINE*

(Received for publication, March 15, 1993, and in revised form, June 7, 1993)

Mitsuhisa Komatsu, Alison M. McDermott, Susan L. Gillison, and Geoffrey W. G. Sharp From the Department of Pharmacology, College of Veterinary Medicine, Cornell University, I t h a , New York 14853

Mastoparan, a tetradecapeptide from wasp venom, stimulated exocytosis in a concentration-dependent manner, which was enhanced by pertussis toxin pretreatment, in the insulin secreting &cell line RINm5F. Mastoparan (3-20 MM) also elevated cytosolic free calcium concentration ([Ca2+]i), a rise that was not atten- uated by nitrendipine. Divalent cation-free Krebs- Ringer bicarbonate (KRB) medium with 0.1 mM EGTA nullified the mastoparan-induced increase in [Ca2+l1, suggesting that the peptide increased Ca2+ influx but not through the L-type voltage-dependent Ca2+ channel. Depletion of the intracellular Ca2+ pool did not affect the mastoparan-induced elevation of [Ca"+]i. Re- markably, in divalent cation-free KRB medium with 0.1 mM EGTA and 2 MM thapsigargin in which mastoparan reduced [Ca2+]i, the mastoparan-stimulated insulin release was similar to that in normal Ca2+-containing KRB medium. Inhibitors of protein kinase C, such as bisindolylmaleimide, staurosporine, and 1-0- hexadecyl-2-0-methyl-rm-glycerol did not suppress the mastoparan-stimulated insulin release. Masto- paran at 10-20 pM did not increase cellular CAMP levels, nor did mastoparan at 5-10 I.LM affect ['Hlara- chidonic acid release. In conclusion, although mastoparan increased [Ca2+Ii, this increase was not involved in the stimulation of insulin release. Rather, the data suggest that mastoparan directly stimulates exocytosis in a Ca'+-independent manner. As GTP-binding proteins (G proteins) are thought to be involved in the process of exocytosis and as mastoparan is known to exert at least some of its effects by activation of G proteins, an action of mastoparan to activate the putative stimulatory G. (exocytosis) protein is likely.

Exocytosis is controlled by a variety of signal transduction systems and their cross-talk. Much information on cellular signaling related to exocytosis has implicated the cytosolic free calcium concentration ( [Ca2+Ii),' CAMP, various phos- pholipids, different protein kinases, and GTP-binding proteins (G proteins) as important components of stimulus- secretion coupling. Although [Ca2+li is believed to play a pivotal role in stimulating exocytosis, and elevation of [Ca2+Ii

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

The abbreviations used are: [Ca2+];, cytosolic free calcium concentration; G protein, GTP-binding protein; PTX, pertussis toxin; TPA, 12-O-tetradecanoylphorbol-13-acetate; AMG-Clc l-0-hexadecyl-2- O-methylglycerol; BHQ, 4-benzohydroquinone; KRB, Krebs-Ringer bicarbonate; IPS, inositol 1,4,5-trisphosphate; GTPyS, guanosine 5'- 3-O-(thio)triphosphate.

induced by secretagogues has been observed in many types of endocrine cells, these results do not mean that the elevation of [Ca2+Ji is indispensable for exocytosis.

Mastoparan, a tetradecapeptide purified from wasp venom (l), is a versatile peptide. Mastoparan stimulates phospholipase A2 (2), phosphoinositide-specific phospholipase C (3-51, DNA synthesis in Swiss 3T3 cells (6), and it binds to calmod- ulin ( 7 ) . Stimulatory (8) or suppressive (9) effects of the peptide on adenylylcyclase have been reported. Mastoparan also acts as a secretagogue in many types of cells including mast cells (I), pituitary lactotrophs (IO), chromaffin cells (11), platelets (12), pulmonary alveolar cells (9), pancreatic @-cells (13, 14), and RINm5F cells (15). In some of these cells (9, 14) and in other cell types (16, 17), mastoparan increases [Ca2+Ii. Recently, Higashijima et al. (18, 19) reported that mastoparan mimicked hormone receptor activity and stimulated guanine nucleotide exchange by heterotrimeric G proteins. A cross-linking study revealed a binding site for mastoparan on the amino terminus of the a-subunits of G proteins (20).

The present study was designed to study a novel mechanism of mastoparan-induced exocytosis, which bypasses the increase in [Ca2+Ii. To delineate the mechanism of this Ca2+- independent mastoparan-induced exocytosis, we examined its effects on insulin release and changes in [Ca2+Ii using the insulin-secreting @-cell line, RINm5F.

EXPERIMENTAL PROCEDURES

Materials-Mastoparan, pertussis toxin (PTX), sulfinpyrazone, nitrendipine, 12-O-tetradecanoylphorbol-13-acetate (TPA), staurosporine, mepacrine, and fura-2 acetoxymethyl ester (fura-a/AM) were from Sigma. Forskolin, bisindolylmaleimide, l-O-hexadecyl-2-0- methylglycerol (AMG-C16), and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ) were from Calbiochem. Cyclic AMP radioimmunoassay kits and [3*P]NAD (30 Ci/mmol) were from Du Pont-New England Nuclear. [3H]Arachidonic acid (208 Ci/mmol) was purchased from Amersham Corp. Thapsigargin was from Research Biochemicals Incorporated (Natick, MA).

Cell Culture for RINm5F-RINm5F cells were maintained in monolayer culture in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 pg/ml streptomycin, and 100 units/ml peni- cillin at 37 "C in a 95% air plus 5% COz atmosphere. Cells a t passages 57-65 were used.

Insulin Release-In static incubation experiments, the cells were grown in 16-mm diameter wells for 3-5 days, and the medium was changed on the day before experiments. Cells were incubated at 37 "C for 30 min in Krebs-Ringer bicarbonate (KRB) buffer containing 129 mM NaCl, 5 mM NaHC03, 4.8 mM KCl, 1.2 mM KHzP04, 1.0 mM CaC12, 1.2 mM MgSO,, 2.8 mM glucose, 0.1% bovine serum albumin, and 10 mM HEPES at pH 7.4. In some experiments, inhibitory agents such as bisindolylmaleimide, staurosporine, and AMG-C16 were present throughout the experiments. The preincubation solutions were replaced with KRB buffer containing test agents, and cells were incubated at 37 "C. The incubation media were sampled at 20 or 30 min after incubation, centrifuged to remove possible contamination

23297

23298 Mastoparan Stimulates Ca2+-independent Insulin Release

by detached cells, and the supernatants used for radioimmunoassay of insulin. Cells were counted after incubation, and the rate of insulin release was expressed as pg/103 cells/2O or 30 min. In perifusion experiments, cells were grown to approximately the same density as those used for the static incubations. Cells dispersed in a Ca2+-free solution with EDTA plus trypsin were loaded in each perifusion chamber (10' cells in 0.7 ml/chamber) and perifused with KRB buffer at 37 "C at a flow rate of 1 ml/min. For Ca2+-free conditions, KRB buffer devoid of CaZ+ and M%+ (divalent cation-free KRB) was used throughout the experiments. EGTA (0.1 mM) and tbapsigargin (2 pM) were added 10 min prior to starting the stimulation with mastoparan and then remained present throughout. The experiments started after a 30-min perifusion equilibration period. Samples were collected every min, and insulin in the perifusate was measured by radioimmunoassay. The rate of insulin release was expressed as pg/ io3 cells/min.

Measurement of Cytosolic-free Ca" Concentration-The [Ca2+]i was fluorometrically measured using the Ca2+-sensitive fluorescent dye fura-2 (21). Cells grown to the same density as those used for the insulin release studies were examined. The cells were dispersed into single cells in a Ca2+-free solution with EDTA plus trypsin. Cells were suspended at 1.5 X 10' cells/ml in RPMI 1640 medium with 5% fetal bovine serum at pH 7.4 and maintained at room temperature prior to loading. For fura-2 loading, the cell suspension was centrifuged, resuspended in KRB buffer containing 0.25 mM sulfinpyrazone with 1 p~ fura-B/AM at 4 X 10' cells/ml and incubated with contin- uous shaking at 37 "C for 30 min. The fura-2-loaded cells thus obtained were washed and resuspended in KRB buffer containing 0.25 mM sulfinpyrazone at 1.5 X 10' cells/ml, and 3 ml of suspension was placed in each quartz cuvette. In some experiments, after loading, divalent cation-free KRB buffer without sulfinpyrazone was used. During experiments and while in the spectrofluorometer (Perkin- Elmer-Cetus Instruments LS-5), the cell suspensions were continu- ously stirred with a small magnetic bar within the cuvettes. The temperature of the cell suspension was maintained at 36-37 "C by circulating warm water through the cuvette holder. An excitation wavelength of 340 nm and an emission wavelength of 510 nm were used to calculate the [Ca2+];. In many experiments, excitation wavelengths of 340 and 380 nm were used in turn by manually shifting the wavelength to monitor changes of both Ca2+-sensitive and -insensitive fluorescence. The [Caz+]i was calculated according to the formula of Tsien and co-workers (21,22), as we have done previously (23, 24).

[Caz+]i = Kd(F - Fmh)/(Fm= - F ) 0%. 1)

K d is the effective dissociation constant for Ca2+ binding to fura-2 (224 nM) (21). F, F-, and F,, are the fluorescences at a time of interest, a t zero and saturated [Ca2+]i, respectively, after correcting for extracellular fura-2 and autofluorescence of the cells. Fluorescence due to extracellular fura-2 was determined by adding manganese to the cells at the beginning (in paired cuvettes) and end of each experiment. Since the rate of fura-2 leakage from the cells was constant throughout an experiment, the extracellular fura-2 at any time in an experiment was obtained by extrapolation. Autofluores- cence of the cells was obtained by the addition of manganese after Triton X-100. The cells were lysed with Triton X-100 followed by the addition of EGTA plus Tris-HC1 to obtain F,, and Fmin, respectively.

Measurement of Cellular CAMP Leuel"RINm5F cells were grown in 16-mm diameter wells for 4-5 days. Cells were preincubated at 37 "C for 15 min with gentle shaking in KRB buffer. The solution was replaced with KRB buffer containing mastoparan (10 or 20 p M ) or forskolin (1 p ~ ) , and cells were incubated at 37 'C for 10 min. At the end of the incubation, the assay buffer was removed and kept at -20 "C until radioimmunoassay for insulin. The reaction was termi- nated by the addition of 0.25 ml of ice-cold 6% trichloroacetic acid to each well. CAMP was quantitated by radioimmunoassay.

Arachidonic Acid Rehase-RINm5F cells were cultured in media containing 0.25 pCi/ml [3H]arachidonic acid for 20 h. Incorporation of isotope into the cells was 40-75%. Cells were washed three times and incubated with 1 ml of KRB buffer containing various concentrations of mastoparan for 20 min. When mepacrine, a phospholipase Az inhibitor, was used, this was added with the mastoparan. After incubation, the media were aspirated and immediately placed on ice in Eppendorf tubes. The media were then centrifuged at 3,000 rpm for 2 min at 4 "C to remove detached cells, and the top 200 pl was

aspirated for scintillation spectrometry. Another 100 pl of the media was kept a t -20 "C until radioimmunoassay for insulin.

Pertussis Toxin Pretreatment and ADP-ribosylation of RINmSF Cell Membranes-PTX pretreatment was carried out during a 20-h culture with 30 ng/ml PTX. For preparation of a crude membrane fraction, the cells were washed twice with ice-cold phosphate-buffered saline and harvested. After lysis in hypotonic Tris, pH 7.4, the samples were centrifuged for 10 min at 1,000 X g. The supernatant was then centrifuged for 30 min at 96,000 X g. The resulting partic- ulate fraction was suspended in 20 mM Tris, pH 7.4, to a concentration of 5-10 pg/ml and stored at -70 "C.

The PTX holoenzyme was activated a t 30 "C for 20 min in 10 mM dithiothreitol, 100 p~ ATP, and 10 mM Tris, pH 7.4. The ADP- ribosylation assay was carried out in a 50-p1 reaction volume at 30 "C for 45 min. Final concentrations in the assay were 20 mM Tris, pH 8.0, 5 mM dithiothreitol, 100 p M GDP, 1 mM MgCl2, 10 p~ NAD, 0.5 pci of [32P]NAD/reaction, 20 pg/ml aprotinin, 10 mM thymidine, 2 pg/ml activated PTX, and 30 pg of membrane protein/reaction. The reaction was stopped with the addition of 15 p1 of concentrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer. The membrane proteins were analyzed by electrophoresis on 10% acrylamide gels, containing acry1amide:bisacrylamide at 37.5:l and 6 M urea (Accurate Chemical). ADP-ribosylated proteins were visualized by autoradiography.

Western blotting (semidry electroblotting) was performed using the Milliblot apparatus (Millipore Corp.). After transferring the proteins to Immobilon (Millipore Corp.), AS-7-reactive proteins were visualized using the chemiluminescence method (ECL kit, Amersham Corp.). The AS-7 antiserum was purchased from Du Pont-New Eng- land Nuclear and is directed against the terminal decapeptide of G,il and Gd2.

Statistical Analysis-Results are presented as means k S.E. Statis- tical analysis was by Student's t test for unpaired data.

RESULTS

Mastoparan-induced Insulin Release: Concentration De- pendence-As shown in Fig. l , mastoparan stimulated insulin release from RINm5F cells in a concentration-dependent manner. The rate of release was almost doubled at 4 pM mastoparan, and it increased further at all concentrations tested. The stimulatory effect of mastoparan was completely reversible, and cell viability was well preserved. After stimulation with the various concentrations of mastoparan, and subsequent washout, basal secretion rates were similar to control, prestimulation values. Furthermore, mastoparan-induced insulin release was temperature-sensitive (10 pM mastoparan-induced insulin release; 37 "C, 45 k 3; 30 "C, 22 f 1 pg/103 cells/20 min; mean * S.E., n = 4, p < 0.01).

Time Course of Mastoparan-induced Insulin Release and Effects of Pertussis Toxin Pretreatment-Mastoparan caused a monophasic increase in the rate of insulin release under perifusion conditions (Fig. 2). Upon exposure to mastoparan, the rate of insulin secretion increased after a delay of 1 min, was almost linear for the next 7 min, after which the rate of release reached its peak value. There followed a 10-12-min plateau before the rate began to decline slowly. The gradual decline in secretion rate continued over the next 30 min until the experiment was discontinued. Pretreatment of the cells for 20 h with 30 ng/ml PTX caused a significant enhancement of the rate of release during the initial 15 min of the stimulation. In these PTX-pretreated cells, the effect of mastoparan occurred faster than in the control cells. For example, from 1 to 8 min after exposure to mastoparan, at which point the rate peaked, insulin was released at more than twice the rate of the stimulated control cells. Subsequently, over the period of declining release, PTX-treated cells secreted insulin at a higher rate than the controls. To confirm that the PTX pretreatment of the cells was sufficient to ADP-ribosylate completely all of the PTX-sensitive G proteins, ADP-ribosylation studies using [32P]NAD were performed on broken cell membrane preparations from the RINm5F cells. In crude

Mastoparan Stimulates ea2+-independent Insulin Release 23299

FIG. 1. Mastoparan-induced insulin release as a function of concentration. Closed circles indicate the rates of insulin release from RINm5F cells incubated with various concentrations of mastoparan for 30 min. After the 30-min stimulation period with mastoparan, the cells were washed twice with KRB buffer, and a further 30-min incubation in basal KRB buffer was performed. The insulin release rates under these conditions are indicated by the open circles. Values represent the mean f S.E. of four determinations.

n c - E

0 0 -

0 1 0 2 4 9 0 4 0

Maatoparan (pU)

10 . i

n " { I 10 pM Martoparan c

FIG. 2. Time course of 10 gM mastoparan-stimulated insulin release from PTX-pretreated and control cells. RINm5F cells were pretreated with 30 ng/ml PTX or vehicle solution for 20 h. Dispersed cells ( lo6 cells) were loaded in a perifusion chamber with a volume of 0.7 ml and perifused with KRB buffer at a flow rate of 1 ml/min. Mastoparan was introduced as indicated in the figure. Values represent the mean

S.E. of three determinations. 0 : I I I I I I

0 1 0 2 0 3 0 4 0 5 0

Time (min)

I 2 3 4

e

FIG. 3. ADP-ribosylation of RINm5F cell membranes by PTX. Membrane protein (30 pg) from control (lanes I and 3 ) or PTX-treated cells (lanes 2 and 4 ) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The samples in lunes I and 2 were subjected to PTX-catalyzed [3ZP]ADP-ribosylation. The proteins were transferred to Immobilon, and the bands were visualized by autoradiography (lanes I and 2 ) or chemiluminescence after incubation with the AS-7 antiserum and horseradish peroxidase-con- jugated second antiserum (lunes 3 and 4 ) .

membrane preparations from control cells, PTX catalyzed the [32P]ADP-ribosylation of proteins in three bands (Fig. 3, lane I ) . Pretreatment of cells with PTX totally prevented any subsequent ADP-ribosylation of these substrate proteins for PTX (Fig. 3, lane 2 ) .

As shown by the accompanying Western blot in lanes 3 and 4 of Fig. 3, treatment with PTX (25) and ADP-ribosylation (26) reduce the mobility of the G protein a-subunits (in this case a i l and ai2). In addition, the gel conditions used here (6 M urea and 10% acrylamide) also decrease the mobility of these substrates relative to the molecular weight markers (27). Thus, the high apparent molecular weight of the 32P-labeled PTX substrates in lane 1 is due to both treatment with the

toxin and the gel conditions. Interestingly, a i l runs as a single band in the control preparation, and as a doublet in the PTX- treated preparation, perhaps indicating multiple forms of the ADP-ribosylated protein.

Mastoparan-induced Changes in [Cu*+/i-The fluorescence signals in response to 10 p~ mastoparan, monitored in a single cuvette using the two excitation wavelengths of 340 and 380 nm, are shown in Fig. 4. Mastoparan produced an increase in emission (510 nm) following excitation at 340 nm, synchronized with a decrease in emission (510 nm) at 380 nm, indicating an elevation of [Ca2+Ii. The resting level of [Ca2+Ii in cells maintained in KRB buffer with 2.8 mM glucose was 85 k 5 nM (n = 40). Upon administration of mastoparan, the Ca2+-sensitive fluorescence following excitation at 340 nm increased quickly and without delay (Fig. 5). Three different patterns of increased [Ca2+], were observed depending upon the concentration of mastoparan used. With 3 and 5 p~ mastoparan, the [Ca2+], rose rapidly to a peak at 20-30 s and then declined. With 3 p~ the [CaZ+li declined to the starting value (Fig. 5A). With 5 p~ the [Ca"], declined to a plateau still elevated above the basal value (Fig. 5B). With 10 p~ mastoparan, there was only a very slight decline after the peak was reached at 20 s, and [Ca2+]; remained at a stable high plateau level (as shown in Fig. 5C). When the cells were exposed to 20 NM mastoparan, the third pattern was seen (Fig. 50) . In this case, a rapid increase in [Ca2+], occurred to a peak at 10 s. This was followed by a decrease and then an

23300 Mastoparun Stimulates Cu2+-independent Insulin Release

oscillating pattern, the magnitude of the oscillations dimin- ishing as the [CaZ+li continued to increase to its highest value and a stable plateau after 90 s of exposure. Sustained elevated levels of [CaZ++li were observed throughout the recordings (up to 15 min) at the higher concentrations of mastoparan (5-20 PM).

Effects of Thapsigargin, BHQ, Ca2+ Removal, and Nitren- dipine on Mastoparan-induced Changes in [Ca2+Ii-In Fig. 6 are shown changes in [Ca2+]i in response to thapsigargin and BHQ, both inhibitors of microsomal Ca2+-ATPase (28-30), and mastoparan. It is known that the administration of thapsigargin or BHQ, by blockade of intracellular Ca2+ uptake,

20-

O 5 10 1 s 1 I I - 10 #“--=“””

380 nm

Tlmo (mln) FIG. 4. Fluorescence changes in response to 10 p M masto-

paran monitored in a single cuvette using two excitation wavelengths at 340 and 380 nm. Emission wavelength was 510 nm. Note that the increase in emission following excitation at 340 nm synchronizes with the decrease in that at 380 nm.

evokes a temporal elevation of [Ca2+]i and subsequent depletion of the inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ pool. Addition of 2 pM thapsigargin to the RINm5F cells induced a prompt increase in [Caz+Ii which peaked at 144 nM after 18 s (Fig. 6A). The [Ca2+Ii then declined over the next 6 min to the basal value. At this point, the addition of 50 PM BHQ caused a slight decrease, not an increase, of [Caz+li. The lack of an increase in [CaZ+li would be in accord with complete blockade of calcium uptake into the stores by the thapsigargin given previously. The slight decrease could be a result of the known effect of BHQ to block Ca2+ uptake across the plasma membrane (29). Furthermore, when a maximal concentration of carbachol was added at this time after 2 KM thapsigargin, no elevation of [Caz+]i was observed (data not shown). Consequently, it could be con- cluded that the intracellular IP3-sensitive calcium stores were depleted. The effect of mastoparan under these conditions is shown in Fig. 6B. When 10 p~ mastoparan was added to the cells, a very prompt increase in [Caz+Ii was observed. Thus the [CaZ+Ji peaked rapidly, fell briefly, and then increased slowly over the next few min. Regardless of the fine details of the changes in [Caz+Ii, mastoparan is fully capable of increasing [Ca2+Ii at a time when intracellular calcium stores are blocked by thapsigargin. Further confirmation of this conclusion was obtained by experiments in which BHQ was used to block uptake into the IP3-sensitive stores and to deplete them of calcium. As shown by the data in Fig. 6C, the addition of 50 PM BHQ, like thapsigargin, caused an increase in [Caz+],. Subsequent addition of thapsigargin was without effect. Nevertheless, as was the case after thapsigargin, mastoparan was still fully capable of increasing [Ca2+Ii (Fig. 6D). These data suggest that the source of Ca2+ for the increase in [Ca2+], is not mobilization of Ca2+ from the IP3-sensitive stores. Consequently, studies were next performed on cells in the absence of extracellular Ca2+.

The cells, after loading with fura-2, were transferred to divalent cation-free KRB buffer (but not containing EGTA),

C. 0.

e

t 0 a

k - 1 rnh

FIG. 5. Effects of mastoparan on [Cas+], as measured by the fluorescence of fura-2. RINm5F cells were loaded with fura-2 as described under “Experimental Procedures.” The effects of mastoparan at four concentrations on [Ca2+]i were determined as monitored by excitation and emission wavelengths of 340 and 510 nm, respectively. In these particular experiments, peak values of elevated [Caz+],, calculated by the equation shown under “Experimental Procedures,” in response to 3, 5, 10, and 20 PM mastoparan were 99, 129, 192, and 311 nM, respectively. The experiments were repeated at least three times with similar results.

Mastoparan Stimulates Ca'+-independent Insulin Release 23301

A

?! 0 7 U

50 PM I

1 - 1 mtn

C. i D.

t

FIG. 6. Effects of thapsigargin and BHQ on [Cas+], in RINmSF cells and on mastoparan-induced changes in [Ca2+]i. RINm5F cells were loaded with fura-2. The fluorescence was monitored using an excitation wavelength of 340 nm and an emission wavelength of 510 nm. Thapsigargin (2 p ~ ) and BHQ (50 p ~ ) were added as indicated by the vertical arrows. The experiments were repeated at least three times with similar results.

placed in the cuvettes, and fluorescence measurements were begun. Shortly thereafter, EGTA was added to the cuvette to a concentration of 0.1 mM to remove the remaining traces of Ca*+. After stabilization of [Ca2+]i the test agents were added to the cuvettes. The addition of EGTA resulted in a prompt decrease in the fluorescence signal, caused primarily to leaked extracellular fura-2 (Fig. 7 , A and B ) . The subsequent addition of 10 p~ mastoparan failed t o increase [Ca2+Ji and resulted in

a sharp, small but brief decrease in [Ca'+], as the [Ca2+]i level was promptly restored to its previous value, followed by a gradually decreasing level of [Ca2+Ii. Thapsigargin was able to increase [Ca2+];, as shown earlier under normal conditions with Ca2+-containing KRB buffer, although the return to previous levels of [Ca2+Ii was more rapid (Fig. 7 A ) . In the experiment shown in Fig. 7B, thapsigargin was added after EGTA and before mastoparan. Thus, by the time that mas-


FIG. 7. Effects of CaZ+ removal and nitrendipine on mastoparan-induced changes in [Cas+],. A and B, effects of 2 pM thapsigargin and 10 p~ mastoparan on [Ca2+Ii in the absence of extracellular CaZ+. EGTA (final concentration 0.1 mM) was administered at the first arrow to the cell suspension in divalent cation-free KRB buffer. C, effect of 1 pM nitrendipine on the basal [Ca"]; and subsequent effect of 10 p~ mastoparan. The experiments were carried out at least three times with similar results.

A

a# 0 C

0 Q,

rp

0 3

t it

toparan was added, the intracellular calcium stores were depleted and unable to participate, as they usually do, in the protection of [Ca2+]; levels. Under these conditions, mastoparan caused a rapid and persistent decrease in [Caz+],, suggesting an action of mastoparan to increase the permeability of the RINm5F cell membrane to Ca". An obvious possibility for the change in permeability is the L-type voltage-dependent Ca2+ channels activatedphysiologically by depolarization with secretagogues such as glucose. To test this possibility, we examined the effect of nitrendipine, an inhibitor of L-type voltage-dependent calcium channels, on mastoparan-induced changes in [Ca"]i. The results of such an experiment, carried out in normal Ca2+-containing KRB buffer, are shown in Fig. 7C. Nitrendipine, at a concentration of 1 FM, caused a small decrease in [Ca2+Ii. However, it failed to prevent the charac- teristic rise in [Ca2+]; in response to mastoparan, suggesting that mastoparan does not cause Ca2+ entry via the L-type Caz+ channels and presumably uses either the T-type channel in these cells (31) or a novel mechanism.

Mastoparan-induced Insulin Release in Ca2+-free Condi- tions-In the absence of extracellular Ca2+, mastoparan failed to increase [Ca2+Ii, and in the absence of extracellular Ca2+ and presence of thapsigargin, mastoparan decreased [Ca2++Ii. The effect of mastoparan to stimulate insulin release was,

A.

0.

C. I therefore, investigated under these conditions. Perifusion experiments using divalent cation-free KRB buffer with 0.1 mM EGTA and 2 p~ thapsigargin were performed to evaluate the time course of mastoparan-induced insulin release in the Ca2+- free and lowered [Ca"]; condition (Fig. 8). To mimic faithfully the condition in which mastoparan reduced [CaZ+li (as indicated in Fig. 7 B ) , the cells were loaded with fura-2/AM as for the measurement of [Ca2+]i. The perifusion experiments started after a 30-min equilibration period. Ten min prior to the administration of mastoparan, 0.1 mM EGTA and 2 ~ L M thapsigargin were added and remained present for the rest of the experiment. As shown in Fig. 8, 10 p~ mastoparan was introduced at 20 min, a time that was equivalent to the 50 min taken for the experiments in which [Ca2+Ii was measured under these conditions and in which mastoparan reduced the [Ca2+Ii level. These perifusion experiments again revealed the marked insulin release response to mastoparan. The time course in this Ca2+-free, thapsigargin-treated condition was similar to that under normal conditions (cf. Fig. 2). Further- more, the total amount of insulin released during 30 min in the Caz+-free condition (Fig. 8) was equivalent to that in normal KRB buffer (cf. Fig. 2).

Effects of Mastoparan on Cellular CAMP Levels-Masto- paran, at 10 and 20 PM, strongly stimulated insulin release

Mastoparan Stimulates Ca2+-independent Insulin Release 23303

but had no effect on cellular cAMP levels (Table I). The cells were capable of raising cAMP because the concentration of cAMP in the cells was doubled by 1 pM forskolin.

Effects of Inhibitors of Protein Kinase C on Mastoparan- induced Insulin Release-The possibility that mastoparan was activating protein kinase C activity was investigated. The effects of the protein kinase C antagonist bisindolylmaleimide (32) on insulin release stimulated by 100 nM TPA and by 16 p~ mastoparan are shown in Fig. 9. The antagonist bisindolylmaleimide reduced TPA-stimulated insulin release by 30% at 0.1 p~ and by 65% at 1.0 p ~ . In contrast, mastoparan- stimulated release was not significantly inhibited, and the rate was reduced by only 17% at 0.1 p ~ , a level of inhibition which remained constant as the concentration of the antagonist was increased to 0.3 and 1.0 p ~ . Basal release of insulin increased by 9% in the presence of 1.0 p~ bisindolylmaleimide, but the increase was not statistically significant. Studies with other inhibitors of protein kinase C also failed to dem- onstrate the involvement of protein kinase C in the action of mastoparan. Staurosporine (100 nM) did not inhibit the 16 p~ mastoparan-induced insulin release (basal release: 12 f 1, mastoparan: 28 * 2, mastoparan plus staurosporine: 28 f 3 pg/103 cells/20 min; n = 4). Another protein kinase C inhibitor, AMG-C16 (120 pM), also did not affect the 16 @f mastoparan-induced insulin release (basal release: 33 f 2, mastoparan: 55 -t 3, mastoparan plus AMG-CI6: 53 f 6 pg/103

8 I EGTA + Thaprigargln 1

- . 0 1 0 2 0 3 0 4 0 5 0

Tlme (mln) FIG. 8. Time course of 10 p~ mastoparan-stimulated insulin

release in the absence of extracellular Cas+. RINm5F cells were loaded with fura-2, placed in the perifusion chambers, and perifused with divalent cation-free KRB buffer. After a 30-min equilibration period, the experiments were started. At 10 min, 0.1 mM EGTA and 2 p~ thapsigargin were introduced and remained present for the rest of the experiments. At 20 min, 10 ptM mastoparan was introduced into the perifusate of the test cells. Closed circles indicate 10 g~ mastoparan-induced stimulation of the rate of insulin release. Control cells (open circles) were without mastoparan. Values represent the mean f S.E. of three determinations.

cells/20 min; n = 4). In contrast, both inhibitors suppressed TPA-stimulated release.

Arachidonic Acid Release in Response to Mastoparan-Low concentrations of mastoparan (5-10 pM) did not stimulate arachidonic acid release (assessed by 3H release from cells preloaded with [3H]arachidonic acid) over a 20-min incubation period, whereas both of these concentrations of mastoparan produced significant insulin release (Fig. 10). Although higher concentrations of mastoparan (20-40 pM) increased 3H release in a concentration-dependent manner, the concentration-response curve was to the right of that for mastoparan-induced insulin release. In additional experiments, 3H release induced by 16 p~ mastoparan was strongly suppressed by 0.1 mM mepacrine, a phospholipase Az inhibitor (33), (16 pM mastoparan: 795 & 185, 16 pM mastoparan plus 0.1 mM mepacrine: 252 -t 63 cpm/106 cells/20 min; mean f S.E., n = 4, p < 0.01), and insulin release measured simultaneously revealed no difference between the two groups (16 p M mastoparan: 87 f 3, 16 p~ mastoparan plus 0.1 mM mepacrine: 84 & 4 pg insulin/103 cells/20 min; mean -t S.E., n = 4).

DISCUSSION

Mastoparan has many diverse actions. These include activation of phospholipase AI (2) and phospholipase C ( 3 4 , elevation of [Ca2+Ii (9, 14, 16, 17), and stimulation of guanine nucleotide exchange by G proteins (18, 19). These are all important components of many signal transduction systems including stimulus-secretion coupling. Although mastoparan stimulates exocytosis in many types of cells (1, 9-15), the precise mechanism of mastoparan-stimulated exocytosis re- mains unknown. In the insulin-secreting /?-cell line, RINm5F, we have shown that mastoparan stimulates insulin release and elevates [Ca2+Ii. Strikingly, this increase in [Ca2+]i is unrelated to the stimulation of insulin secretion. Because of this and because mastoparan has multiple actions on cell membranes (34-38) and intracellular components (7, 39), we have examined the nature of the mastoparan-induced stimulation of insulin release.

In the RINm5F cells, the effect of the peptide, at least at concentrations up to 20 pM, is caused by stimulated exocytosis, not nonspecific cellular destruction. We list the following as the basis for this conclusion. 1) Cell numbers and their gross microscopic appearance after stimulation with various concentrations of mastoparan up to 20 p~ showed no significant changes. Higher concentrations of the peptide (230 p ~ ) sometimes decreased the attached cell numbers after static incubation. 2) Basal insulin secretion, after exposure to mastoparan and subsequent removal by washing, was the same as the control cells. 3) Cell numbers and the insulin release responses to 10 p~ mastoparan, 100 nM TPA, or 10 p~ forskolin did not change even after pretreatment for 20 h with 5 pM mastoparan (data not shown). 4) Lowering the ambient temperature to 30 “C greatly suppressed 10 p~ mastoparan-

TABLE I Effects of mastoparan and forskolin on cellular CAMP level and insulin release

RINm5F cells were maintained in 16-mm diameter wells for 4-5 days. After 15 min of preincubation in KRB buffer, the cells were incubated with 10 and 20 p~ mastoparan or 1 PM forskolin at 37 “C for 10 min with gentle shaking. Cellular cAMP and released insulin were determined using radioimmunoassay as described under “Experimental Procedures.” Values are mean k S.E. from a single experiment performed in triplicate.

~~~~~

Mastoparan Forskolin 0 10 20 (1 lrMf

N.U

cAMP level (fmol/gg protein) 20.5 k 3.5 23.8 k 1.9 20.1 k 1.1 Insulin release (pg/pg protein/lO min) 126 & 10

40.7 & 5.2 417 k 57 475 k 57 103 * 5


E - carbd

0 0 . 2 0.4 0 - 6 0 . 0 1 .o

Bisindolylmalelmide (pM) FIG. 9. Effects of various concentrations of bisindolylmal-

eimide on 16 pM mastoparan- and on 100 nM TPA-induced insulin release in RINm5F cells. Static incubations for 20 min, as described under "Experimental Procedures," were performed. The amounts of insulin released by 16 pM mastoparan or by 100 nM TPA in the absence of bisindolylmaleimide were expressed as 100%. Changes in insulin release was then expressed as a percent of the stimulated insulin release. For clarity, basal insulin release was expressed as 0%. Absolute values of insulin release were 24 & 1 pg/103 cells/20 min for the basal; 45 5 2 pg/103 cells/20 min for the mastoparan-stimulated; and 98 +. 6 pg/103 cells/20 min for the TPA- stimulated. Values represent the mean & S.E. of four determinations. ** indicates p < 0.01 uersus the control value.

400 . . 3

s d

C 0

ap - [3H]M R o k 8 r

I I I I I 0 1 0 2 0 3 0 4 0

Mastoparan (pM) FIG. 10. Effects of mastoparan on ['HI and insulin release

in RINm5F cells, preloaded with ['HJarachidonic acid. RINm5F cells were loaded with 0.25 pCi/ml [3H]arachidonic acid ( ? H I M ) in culture media for 20 h. Cells were washed three times with KRB buffer. Then 20-min incubations with various concentrations of mastoparan were carried out. Released radioactivity was counted by liquid scintillation spectrometry. Radioimmunoassay for insulin was also performed. Both 3H release and insulin release were expressed as percent response (mean values in the absence of mastoparan were considered 100%). The absolute value of basal insulin release was 31 -+ 2 pg/103 cells/20 min. Values indicate the mean f S.E. of four determinations. * indicates p < 0.05; ** indicatesp < 0.01 uersus the control values.

induced insulin release, suggesting that temperature-dependent exocytosis, rather than nonspecific membrane perturba- tion, was taking place.

As to the mechanisms underlying the mastoparan-induced insulin release, we have excluded the involvement of cAMP and protein kinase A because no effect of mastoparan to increase cAMP content was detected in RINm5F cells. Pro- tein kinase C, another important component in signal transduction, was also considered. Bisindolylmaleimide, a specific

and potent inhibitor of protein kinase C, failed to suppress the insulin release induced by mastoparan, although it suc- cessfully suppressed that induced by TPA. Furthermore, other types of protein kinase C inhibitors including staurosporine (40) and AMG-CIG (41) did not inhibit insulin release in response to mastoparan. These results suggest that protein kinase C, like PKA, is not involved in the mechanism of mastoparan-induced insulin release in RINm5F cells.

Activation of phospholipase AZ with subsequent arachidonic acid release was also a candidate player in mastoparan-induced insulin release because mastoparan is known to stimulate phospholipase AB in cell-free systems (2) and to stimulate arachidonic acid release (9, 12). In RINm5F cells, mastoparan caused a concentration-dependent activation of arachidonic acid release. However, its concentration dependence was clearly to the right relative to that for the stimulation of insulin release. Additionally, mepacrine, a known inhibitor of phospholipase AZ, did not suppress mastoparan-stimulated insulin release. These results rule out a role for phospholipase AZ activation in mastoparan-induced insulin release, although it is possible that at higher concentrations of the peptide, activation of phospholipase Az may contribute to mastoparan- induced insulin release.

IP3-induced mobilization of intracellular Caz+ is reported to be the cause of mastoparan-induced [Ca"]; elevation (9, 14), although in neutrophils, both intra- and extra-CaZ+ pools participated in the elevation of [Ca2+]i (16). In RINmSF cells, the Ips-sensitive intracellular Ca2+ pool is not involved in the mastoparan-induced [Ca2+]i elevation. Instead, the elevation of [Ca"]i was caused by increased Ca2+ influx, but not appar- ently through the L-type voltage-dependent calcium channels. This is suggested by the failure of nitrendipine at a super- maximal concentration to block the rise in [Ca'+];. However, this conclusion needs confirmation by direct electrophysiolog- ical characterization of the Ca2+ currents induced by mastoparan. It is conceivable that the L-type Caz+ channels are rendered insensitive to nitrendipine and other dihydropyri- dines by the action of mastoparan. It is known that mastoparan causes increased membrane permeability (37, 38) but, the nature of the entry path for Ca2+ detected here is not known. The mastoparan-induced decrease in [Ca2+]i seen in the absence of extracellular Ca2+ is presumed to be caused by accelerated efflux of Ca2+ resulting from the increased membrane permeability. This change in permeability causes increased influx of Ca2+ down the electrochemical gradient in the presence of extracellular Ca", but efflux in its absence. The power and speed of the intracellular Ca2+ stores to correct changes in [Caz+]; rapidly can be inferred from the data shown in Fig. 7. It can be seen in Fig. 7A that mastoparan causes a rapid drop in [Ca2'Ji which, in less than 20 s, is reversed to the level recorded just prior to exposure to mastoparan. In contrast, when the Ca2+ stores have been depleted by treatment with thapsigargin (Fig. 7 B ) , exposure to mastoparan results in a rapid and profound decrease in [Ca"]; which is not corrected.

Remarkably, the stimulation of insulin release by mastoparan was unchanged by the increased [Caz+li seen in normal KRB medium or by the decreased [Ca2+]; seen with mastoparan in the presence of EGTA and thapsigargin. Thus there is no functional linkage between [Ca2+Ii and exocytosis in mastoparan-stimulated insulin release. In view of this dissociation, what are the mechanisms of mastoparan-induced insulin release? Barrowman et al. (42) reported that in permeabilized neutrophils, a nonhydrolyzable analog of GTP, GTP+, could activate the secretory process by actions at two distinct locations: one was at the level of the receptor involv-

Mastoparan Stimulates Ca2+-independent Insulin Release 23305

ing the activation of PI hydrolysis, and the other involved direct activation of the exocytotic mechanism. Similarly, GTPrS stimulated Caz+-independent, PTX-insensitive insulin release from permeabilized RINm5F cells (43). Gomperts (44) coined the term “G, proteins” as the putative G proteins directly coupled with exocytosis. It is possible to speculate, therefore, that mastoparan directly activates the putative stimulatory G, protein(s), which is (are) insensitive to PTX pretreatment and which stimulate(s) exocytosis beyond the elevation of [Ca2+]i. Because the action of mastoparan is exerted at a point beyond the effective site of Ca2+ in stimulus- secretion coupling, it bypasses this site and effectively “takes over” the signaling pathway. Thus, an increase or decrease in [Ca2+Ii is without effect upon mastoparan-stimulated exocytosis.

Investigation of the “distal” mechanisms in the control of exocytosis have only recently begun to clarify the biochemistry involved, although the existence of stimulatory control points beyond the Ca2+ dependence has been recognized for several years. Thus TPA in the RINm5F cell stimulates insulin secretion in the absence of extracellular Ca2+ and decreased [Ca2+Ii, indicating a role for protein kinase C late in stimulus-secretion coupling and beyond the site of Ca2+ action (23). Nonhydrolyzable analogs of GTP stimulate exocytosis in the presence of exceedingly low concentrations of Ca2+ in permeabilized /I-cells (43) thus confirming what is believed now from similar data in several cell types that G proteins are involved at a late step (45-47). More recently, it has been shown that the synthetic peptide related to the effector domain of Rab 3a is capable of causing exocytosis, implicating this or a related small G protein in the mechanisms leading to exocytosis (48-50). In view of these data, the involvement of small G proteins in mammalian cell exocytosis seems likely. However, the involvement of large G or heterotrimeric G proteins in the control of exocytosis also seems probable. Inhibition by galanin of the Ca’+-independent stimulation of insulin release by TPA was demonstrated and found to be sensitive to PTX. Almost certainly this indicates heterotrimeric G protein involvement at a distal site. The enhancement of mastoparan-stimulated insulin secretion in PTX-treated cells is in line with an action of heterotrimeric G proteins to inhibit exocytosis a t a late stage. The action of PTX in this situation is sufficient to relieve the inhibition and therefore increase the secretion rate. It seems possible that activation of multiple G proteins in the cells by mastoparan results in a variety of actions determined by the cell and its G protein composition. In the RINm5F cells, and others, the net effect of both stimulatory and inhibitory influences is a stimulation of exocytosis. This may lead to an explanation, eventually, of the reports that mastoparan-induced exocytosis in different cell types can be both sensitive and insensitive to PTX.

Possible involvement of the putative exocytosis-linked G, proteins in mastoparan-induced exocytosis was reported in ATP-permeabilized mast cells (46) and platelets (8). Although no information about PTX sensitivity is available from the study of the platelets (8), in ATP-permeabilized mast cells their putative stimulatory G. proteins were sensitive to PTX pretreatment. Using chromaffin cells, Sontag et al. (47) indicated that PTX-sensitive inhibitory G, proteins might be coupled with the final stages of the secretory process. Also, in RINm5F cells, the inhibitory G, proteins that are sensitive to PTX pretreatment appear to be coupled with the final stages of exocytosis because PTX pretreatment significantly poten- tiated the mastoparan-induced insulin release from the RINm5F cells in our perifusion experiments.

In conclusion, mastoparan stimulated Ca2+ influx, increased [Ca2+Ii, and stimulated insulin secretion in the RINm5F cell. Mastoparan had no effect on cellular CAMP levels, thus excluding a role for protein kinase A. The effect of mastoparan was not affected by inhibitors of protein kinase C, thus excluding this kinase also. A role for phospholipase A2 activation was eliminated by comparison of the effects of mastoparan and of mepacrine on arachidonic acid release and insulin release. Remarkably, the effects of mastoparan on Ca2+ handling and [Ca2+Ii were not involved in the mechanism(s) by which mastoparan stimulated insulin secretion. Rather, the stimulatory effect was found to be exerted on the exocytotic process per se or close to it in the final stages of stimulus-secretion coupling. This “late” action of mastoparan bypasses the site at which Ca2+ normally exerts its effect and, as a consequence, preempts any effect of Ca2+. This was demonstrated by the fact that neither an increase nor a decrease in [Ca2+Ii had any effect upon the ability of mastoparan to stimulate release. As G proteins are thought to be involved in the process of exocytosis and as mastoparan is known to exert at least some of its effects by activation of G proteins, an action of mastoparan to activate the putative stimulatory G. protein is likely.

Acknowledgments-We thank T. Seaman and X. H. Tao for expert technical assistance and for culture of cells.

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Mastoparan Stimulates Exocytosis at a Ca2+-independent Late Site