J Biol Chem, Vol. 274, Issue 34, 23901-23909, August 20, 1999

Multiple Protein Kinase Pathways Are Involved in Gastrin-releasing Peptide Receptor-regulated Secretion^*

Mark R. Hellmich^§, Kirk L. Ives, Vidyavathi Udupi, Melvyn S. Soloff^¶, George H. Greeley Jr., Burgess N. Christensen, and Courtney M. Townsend Jr.

From the Departments of  Surgery,  Physiology and Biophysics, and ^¶ Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas 77555

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Gastrin-releasing peptide (GRP) and its amphibian homolog, bombesin, are potent secretogogues in mammals. We determined the roles of intracellular free Ca²⁺ ([Ca²⁺]_i), protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) in GRP receptor (GRP-R)-regulated secretion. Bombesin induced either [Ca²⁺]_i oscillations or a biphasic elevation in [Ca²⁺]_i. The biphasic response was associated with peptide secretion. Receptor-activated secretion was blocked by removal of extracellular Ca²⁺, by chelation of [Ca²⁺]_i, and by treatment with inhibitors of phospholipase C, conventional PKC isozymes, and MAPK kinase (MEK). Agonist-induced increases in [Ca²⁺]_i were also inhibited by dominant negative MEK-1 and the MEK inhibitor, PD89059, but not by an inhibitor of PKC. Direct activation of PKC by a phorbol ester activated MAPK and stimulated peptide secretion without a concomitant increase in [Ca²⁺]_i. Inhibition of MEK blocked both bombesin- and phorbol 12-myristate 13-acetate-induced secretion. GRP-R-regulated secretion is initiated by an increase in [Ca²⁺]_i; however, elevated [Ca²⁺]_i is insufficient to stimulate secretion in the absence of activation of PKC and the downstream MEK/MAPK pathways. We demonstrated that the activity of MEK is important for maintaining elevated [Ca²⁺]_i levels induced by GRP-R activation, suggesting that MEK may affect receptor-regulated secretion by modulating the activity of Ca²⁺-sensitive PKC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bombesin (BBS)¹-like peptides are distributed throughout the central nervous system and gastrointestinal tract of mammals, where they modulate metabolism, behavior (1), smooth muscle contractility (2), chemotaxis (3), and exocrine and endocrine processes. Many of the biological effects attributed to the amphibian peptide, BBS, and its mammalian homolog gastrin-releasing peptide (GRP) are a consequence of their potent secretory activity and occur secondarily to the release of other peptide hormones. For example, in the small intestine, the effects of BBS on smooth muscle contraction are partially due to BBS-induced secretion of motilin from intestinal M-cells (4). In isolated muscle strips from the lower esophageal sphincter of rabbits, BBS-induced muscle contractions are blocked by substance-P receptor antagonists, suggesting that substance-P is the direct mediator of contractions in this preparation (5). In the stomach, BBS stimulates gastric acid secretion from parietal cells indirectly by stimulating gastrin release from stomach G-cells (6, 7). BBS-stimulated secretion also plays a role in the proliferation of some tumors of the lung (8) and stomach (9) by participating in autocrine and/or paracrine growth loops. Despite the important role of BBS-stimulated secretion in many normal and pathophysiological processes, little is known about the intracellular signal transduction pathways regulating this activity.

A family of G-protein-coupled receptors mediates the actions of BBS. Three BBS receptor subtypes have been cloned and characterized in humans: 1) gastrin-releasing peptide (GRP)-preferring receptors (GRP-R), 2) neuromedin B-preferring receptors, and 3) the bombesin receptor subtype 3 (10). Agonist binding to GRP-R stimulates phospholipase C- (PLC-) resulting in the production of inositol 1,4,5-trisphosphate and diacylglycerol (DAG), an increase in the concentration of free cytosolic Ca²⁺ ([Ca²⁺]_i), and the activation of both protein kinase C (PKC) (11-13) and mitogen-activated protein (MAP) kinase pathways (14).

To investigate the role of these pathways in BBS-induced peptide secretion, we developed a human neuroendocrine-like cell line that expresses recombinant human GRP-R, called BON/GRP-R. The parental BON cell line was derived from a human metastatic carcinoma tumor of the pancreas (15). BON and BON/GRP-R cells exhibit morphological and biochemical characteristics consistent with the phenotype of a neuroendocrine cell, including the presence of numerous dense-core granules and the expression and secretion of chromogranin-A (CGA), neurotensin (NT), serotonin, and pancreastatin (16, 17). Unlike primary cultures of canine gastric G-cells, which have been used by others to examine BBS-induced secretion (7), BON/GRP-R can be maintained in long term culture without noticeable changes in their secretory activity. In addition, they have a lower level of constitutive or spontaneous peptide release compared with the glucagonoma cell line, STC-1. Using BON/GRP-R cells and a combination of single cell calcium imaging, whole cell voltage clamp, radioimmunoassay (RIA), and the reverse hemolytic plaque assay (RHPA), we have determined the roles of agonist-induced increases in [Ca²⁺]_i, PKC, and MAPK pathways in GRP-R-regulated exocytosis. We have found that GRP-R-activated secretion requires a sustained elevation in [Ca²⁺]_i that is initiated by Ca²⁺ release from intracellular stores and maintained by Ca²⁺ influx across the plasma membrane and through the activity of MEK-mediated pathways. However, an agonist-induced increase in [Ca²⁺]_i will not stimulate secretion without activation of PKC and the subsequent activation of downstream MAPK/ERK. MEK regulation of [Ca²⁺]_i suggests that this kinase pathway may affect receptor-regulated secretion, in part, by modulating the activity of Ca²⁺-sensitive PKC through a feedback loop mechanism.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- BON cells, stably transfected with a human gastrin-releasing peptide receptor cDNA (GRP-R), were grown at 37 °C in a humidified atmosphere of 95% air and 5% CO₂ in DMEM/F12K (1:1) medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) and Geneticin (G-418, 400 µg/ml).

Calcium Imaging-- Real-time recording of [Ca ²⁺]_i was performed in single BON/GRP-R cells using methods described previously (18). Cells were plated on glass coverslips (25 mm) at a density of approximately 1.5-3 × 10⁵ cells/coverslip, cultured for 48 h, washed with KRH (25 mM HEPES, pH 7.4, 125 mM NaCl, 5 mM KCl, 1.2 KH₂PO₄, 1.2 mM MgSO₄, 2 mM CaCl₂, 6 mM glucose), and loaded with 2 µM fura-2AM (Molecular Probes, Eugene, OR) for 50 min at 25 °C. Loaded cells were washed three times with KRH and incubated in KRH plus 0.1% bovine serum albumin for 60 min at 25 °C in the dark. Cells were challenged with various concentrations of BBS and imaged using a Nikon Diaphot inverted microscope (Garden City, NY). The microscope was coupled to a dual monochromator system via a fiber optic cable (Photon Technology International (PTI), South Brunswick, NJ). Fluorescence was detected using an intensified charged coupled device camera (Dage-MTI, Inc., Michigan City, IN) and images processed using ImageMaster software (PTI).

RIA-- BON/GRP-R cells (5 × 10⁵ cells/well) were plated into six-well culture plates (Falcon Labware) in culture medium consisting of DMEM/F12K (1:1) supplemented with 5% FBS and 400 µg/ml G-418. After 48 h, cells (8 × 10⁵ cells/well) were washed with phosphate-buffered saline and treated with bombesin (BBS) (10¹⁰ M to 10⁶ M), diluted in KRH, at room temperature. Following BBS treatment, the NT or CGA content of the medium was determined by RIA as described previously (19, 20). The NT antiserum was generated in rabbits using full-length porcine NT as antigen. CGA antiserum was produced in rabbits using synthetic rat CGA (amino acid residues 359-389) linked to bovine serum albumin as antigen. The sensitivities and ID₅₀ values for the NT RIA and CGA RIA were 100 pg/ml and 1 ng/ml and 40 fmol/ml and 0.4 pmol/ml, respectively.

RHPA-- RHPA assays were preformed as described previously (21) and modified to allow the simultaneous measurement of [Ca²⁺]_i with fura-2 (22). Cells were cultured, loaded with fura-2AM and washed as described above (see "Calcium Imaging"). Before making [Ca²⁺]_i measurements, BON/GRP-R cells were overlaid with solution containing 2% ovine red blood cells conjugated to protein A and anti-CGA antiserum. Single-cell fluorescence measurements were obtained from 20-40 cells/coverslip. Averaged image frames were acquired every 1-8 s, and graphical representations of single-cell ratio values (340/380 nm) were generated using ImageMaster software (PTI). After [Ca²⁺]_i measurements were complete, complement was added to the cells and cells were incubated at room temperature for 1-2 h. Plaque formation was recorded by photographing cells at 0, 1, and 2 h after addition of complement. Data comparing the effects of various pharmacological agents or a dominant negative mutant of MEK-1 were expressed as a percentage of the number of plaques formed by treatment with BBS for 1 h ± S.E. from at least three separate experiments. For all experiments, 600-1000 cells were counted per treatment group. Approximately 3% of the cells exhibited spontaneous peptide secretion. Stimulated secretion was detected in 17-20% of the cells. For evaluation of the effect of various treatments on secretion, statistical analysis was performed using a one-way analysis of variance and Tukey-Kramer multiple comparison test. Statistical significance was assumed for a P value of <0.01.

Electrophysiology-- Cells growing on coverslips were mounted in a Leiden chamber and positioned on the stage of an inverted Nikon Diaphot phase contrast microscope. Patch pipettes were pulled from 1.65-mm (outer diameter) borosilicate glass on a Sutter Instruments P87 horizontal puller. Pipette DC resistance measured 5-7 megohms. Recordings were made using an Axon Instruments Axopatch 200A amplifier, and data were collected using Pclamp (version 6; Axon Instruments). Internal solution consisted of 140 mM KC1, 2 mM MgCl₂, 0.2 mM CaCl₂, 2 mM BAPTA, 11 mM HEPES, pH 7.4, with KOH. Drugs were applied to single cells using a Picospritzer (General Valve Corp.). Changes in membrane potential necessary to activate voltage-dependent conductances were achieved by either step depolarizations or ramp protocols.

Preparation of Cell Extracts and Western Blotting-- BON/GRP-R cells were plated onto 100-mm culture dishes (1 × 10⁶ cells/plate) in DMEM/F12K (1:1) supplemented with 5% heat-inactivated FBS and 400 µg/ml G-418. Two days later, cells were pretreated with various agents and then stimulated with BBS for 5 min. The media were removed by decanting, and the cells were lysed with 1 ml of lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40 detergent, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithioerythritol, 1 mM sodium orthovanadate), scraped, and incubated on ice for 1 h. Non-soluble cell material was removed by centrifugation at 10,000 rpm for 15 min. The protein content of the supernatant was determined by the Bradford method using bovine serum albumin as a standard. Cell extracts (10 µg of protein) were resolved on 10% polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride (Millipore, Milford, MA) membranes as described previously (23). polyvinylidene difluoride membranes were blocked for 2 h with 5% dried skimmed milk in Tris-buffered saline (TBS) (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) and then incubated for 3 h with anti-active MAPK antibody (pTEpY) (Promega, Madison, WI) diluted 1:4000 in 5% milk/TBS. Between primary and secondary antibodies, membranes were washed three times with TBS plus 0.05% Tween 20 detergent. The secondary antibody was a donkey anti-rabbit IgG coupled to horseradish peroxidase and diluted 1:5000 in 5% milk/TBS. Specific immunostaining was detected using a chemiluminescent detection reagent (Amersham Pharmacia Biotech).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effects of BBS Stimulation on [Ca²⁺]_i-- BBS stimulation of BON/GRP-R cells generally produced two distinct [Ca²⁺]_i responses: biphasic or oscillatory. The frequency with which each calcium response was observed depended partially on the concentration of BBS (Table I). Greater than 99% of the cells responded to BBS stimulation at all concentrations tested. At high concentrations of BBS (10⁸ M), 100% of the responding cells displayed a biphasic calcium response (Fig. 1A) characterized by an initial rapid increase in [Ca²⁺]_i followed by a second sustained elevation in [Ca²⁺]_i that slowly declined to resting levels over a period of 10-20 min. The predominant calcium response at lower concentrations of BBS (10¹⁰ M) was an oscillatory increase in [Ca²⁺]_i lasting approximately 15-30 s with a spike frequency ranging from 0.4 to 1.3 transients per min (Fig. 1B). Between each transient, [Ca²⁺]_i generally returned to a level slightly elevated above resting levels. BBS stimulation also induced a single [Ca²⁺]_i spike in a small percentage of cells (2-8%) (Fig. 1C). Like the oscillatory response, the single spike response only occurred at lower concentrations of BBS (Table I).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Three types of [Ca2+]i responses are induced by BBS stimulation of BON/GRP-R19 cells: biphasic (A), base-line oscillations (B), and single spike (C). Each tracing is from a representative single cell. The change in [Ca2+]i is expressed as the ratio of fura-2 fluorescence at 340/380 nm.

Effects of Extracellular Ca²⁺ on BBS-induced Changes in [Ca²⁺]_i-- Both the biphasic and oscillatory calcium responses were dependent on the presence of extracellular Ca²⁺. Replacing the normal KRH solution with a solution that did not contain added Ca²⁺ and included 1 mM EGTA terminated the biphasic and oscillatory [Ca²⁺]_i responses (Fig. 2, A and B). The biphasic response was immediately attenuated following removal of extracellular Ca²⁺, whereas oscillating cells showed a slowing of the spike frequency, followed by complete cessation of the calcium response within 10 min.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Effects of extracellular Ca2+ and La3+ on BBS-induced increases in [Ca2+]i. Replacing the bath solution with KRH without Ca2+ and containing 1 mM EGTA abolished the BBS-induce biphasic (A) and oscillatory (B) [Ca2+]i responses. Addition of 0.2 mM La3+ to cells bathed in normal KRH caused a slowing of spike frequency (C), whereas addition of 1 mM La3+ completely blocked oscillation in [Ca2+]i (D).

Addition of lanthanum (La³⁺) blocked agonist-induced [Ca²⁺]_i oscillations in a concentration-dependent fashion (Fig. 2, C and D). Lanthanum blocks Ca²⁺ influx through the plasma membrane (24) and inhibits the activity of the plasma membrane Ca²⁺-dependent ATPase (25). It is also the most potent metal ion to block the sodium-calcium exchanger in vesicle systems (26) and in the squid axon (27). A low concentration of La³⁺ (0.2 mM) caused a slowing of the spike frequency, whereas a higher concentration (1.0 mM) completely abolished BBS-induced increases in [Ca²⁺]_i.

Whole cell voltage clamp experiments with BON/GRP-R cells revealed an inward current that was activated near 40 mV and peaked near 5 mV (data not shown). This current was slowly inactivating and similar to that seen during activation of L-type voltage-gated Ca²⁺channels. These channels, however, are unlikely candidates for agonist-induced Ca²⁺ influx because BBS failed to depolarize BON/GRP-R cells, a requirement for activation of voltage-gated Ca²⁺ channels. There was no effect of BBS application on BON/GRP-R cell membrane conductance compared with measurements just before and after agonist stimulation. Consistent with these experiments, nifedipine, a blocker of L-type voltage-gated Ca²⁺ channels, did not affect either BBS-induced [Ca²⁺]_i oscillation or biphasic responses (data not shown).

BBS Stimulates Peptide Secretion from BON/GRP-R Cells-- In the presence of extracellular Ca²⁺ (2 mM), maximum release of CGA and NT occurred at BBS concentrations of 10⁸ M and above (Fig. 3, A and B, closed circles). In the absence of extracellular Ca²⁺, BBS failed to stimulate detectable release of either CGA or NT at all concentrations of agonist tested (Fig. 3, A and B, open circles). Addition of 10⁷ M BBS caused a time-dependent increase in CGA and NT secretion that reached a maximum at approximately 30 and 15 min, respectively (Fig. 3, C and D). The time course of peptide release was consistent with the long duration of biphasic calcium response observed at higher concentrations of BBS. In contrast, treatment with a low concentration of BBS (10¹⁰ M), which induced predominantly [Ca²⁺]_i oscillations (Table I), produced only a small increase in NT secretion (Fig. 3D, open circles).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Concentration response and time course of BBS-induced secretion. BON/GRP-R19 cells were incubated for 1 h at room temperature with increasing concentrations of BBS. BBS induced a concentration-dependent increase in CGA (A) and NT (B) release in the presence of extracellular Ca2+ (2 mM) (closed circles). In the absence of extracellular Ca2+, there was no detectable secretion of either CGA (A) or NT (B) (open circles). Cells were treated with BBS (107 M) at room temperature for various lengths of time. CGA (C) and NT (D) secretion was measured by RIA. BBS (107 M) induced a time-dependent release of both peptides (closed circles). At 1010 M BBS, there was minimal release of NT (D, open circles). Each point represents the mean ± S.E. (n = 6) from three separate experiments.

To investigate the relationship between specific patterns of change in [Ca²⁺]_i and peptide secretion from individual cells, we used a combination of fura-2 imaging and RHPA. BON/GRP-R cells were stimulated with 10¹⁰ M BBS; intracellular Ca²⁺ oscillations were recorded in 152 individual cells. When we compared [Ca²⁺]_i records with the results of the RHPA, we found no detectable secretion of CGA from cells exhibiting [Ca²⁺]_i oscillation up to 2 h after stimulation (Fig. 4A). When [Ca²⁺]_i records from individual cells stimulated with 10⁷ M BBS were compared with RHPA data, all cells that developed plaques (about 20% of the total cells) exhibited a biphasic [Ca²⁺]_i response (Fig. 4B, n = 79). Similar results were obtained when NT secretion was compared with [Ca²⁺]_i (data not shown).

View larger version (72K): [in this window] [in a new window]   Fig. 4.   Combined single cell measurements of [Ca2+]i and secretion. BON/GRP-R19 cells, loaded with fura-2, were overlaid with a solution containing 2% ovine red blood cells conjugated to protein-A and anti-CGA antiserum and stimulated with BBS. Agonist-induced [Ca2+]i responses were measured as described under "Experimental Procedures." After calcium recordings were completed, complement was added and cells were incubated for 1-2 h at room temperature to allow for plaque development. A, a representative cell displaying calcium oscillations that did not develop a plaque; B, a representative cell with a biphasic [Ca2+]i response and the developed plaque.

To determine whether cells exhibiting [Ca²⁺]_i oscillations were secretion-competent, we identified oscillating cells and showed that, after 1 h of continuous exposure to BBS (10¹⁰ M), there was no plaque formation by RHPA. When cells were restimulated with 10⁷ M BBS and incubated an additional 1 h, plaques formed around the cells previously exhibiting [Ca²⁺]_i oscillations, indicating that these cells were capable of peptide secretion in the presence of sufficient agonist (data not shown).

GRP-R-regulated Secretion Is Initiated by IP₃-induced Ca²⁺ Release from Intracellular Stores-- Pretreatment of BON/GRP-R19 cells with either the PLC inhibitor, U73122 (1 µM), or the acetoxymethyl ester form of the Ca²⁺ chelator, BAPTA (30 µM), completely blocked BBS-stimulated increases in [Ca²⁺]_i (Fig. 5A) and peptide secretion (Fig. 5B). These data demonstrate that BBS-stimulated peptide secretion is dependent on an increase in [Ca²⁺]_i, which is initiated by an inositol 1,4,5-trisphosphate-induced Ca²⁺ release from intracellular stores.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Effects of a PLC- inhibitor, U73122, and the acetoxymethyl ester form of the Ca2+ chelator, BAPTA, on BBS-stimulated changes in [Ca2+]i (A) and CGA secretion (B). BON/GRP-R19 cells were pretreated for 4-5 min with either 1 µM U73122 or 30 µM BAPTA and stimulated with BBS (107 M). Calcium tracings are the average 340/380 nm ratios from 20-40 individual cells ± S.D. Secretion is expressed as a percentage of plaque-forming cells in 1 h ± S.E. from three separate experiments. Six hundred to 1000 cells were counted per treatment group. Baseline or background secretion from cells treated with Me2SO (DMSO, 0.5%) was approximately 3% of the total cells counted. BBS-induced peptide secretion from 17-20% of the cells. * p < 0.01 versus 0.5% Me2SO.

To determine whether Ca²⁺ released from intracellular stores was sufficient to stimulate secretion, we treated BON/GRP-R19 cells with thapsigargin. Thapsigargin is an irreversible inhibitor of the microsomal Ca²⁺-ATPase reuptake pump that is responsible for maintaining intracellular Ca²⁺ stores. Application of thapsigargin to BON/GRP-R cells in the absence of extracellular cellular Ca²⁺ induced a transient increase in [Ca²⁺]_i (Fig. 6A, Ca²⁺-free) and did not stimulate secretion (Fig. 6B, Thaps (Ca²⁺ free). Bombesin stimulation (10⁷ M), in the absence of extracellular Ca²⁺, produced a similar [Ca²⁺]_i profile and also failed to induce secretion (data not shown). In contrast, thapsigargin treatment of cells bathed in 2 mM extracellular Ca²⁺ induced both a sustained increase in [Ca²⁺]_i (Fig. 6A, 2 mM Ca²⁺) and CGA secretion (Fig. 6B, 2 mM Ca²⁺). These data demonstrate that release of Ca²⁺ from intracellular stores, in the absence of an influx of extracellular Ca²⁺, is insufficient to stimulate peptide secretion. To further evaluate the role of store-released Ca²⁺ in GRP-R-activated secretion, BON/GRP-R cells were pretreated with thapsigargin for 10 min in Ca²⁺-free media in order to deplete the intracellular Ca²⁺ stores. Following thapsigargin treatment, the bath solution was replaced with KRH containing 2 mM Ca²⁺ and after a 20-min recovery, the cells were overlaid with ovine red blood cells and stimulated with BBS (10⁷ M). Fig. 6C shows the effects of thapsigargin pretreatment, addition of extracellular Ca²⁺, and BBS stimulation on the [Ca²⁺]_i record. Bombesin failed to induce either an increase in [Ca²⁺]_i (Fig. 6C) or CGA secretion (Fig. 6B, Thaps + BBS) from cells in which the intracellular Ca²⁺ stores were depleted by pretreatment with thapsigargin. Together, these data suggest that GRP-R-mediated Ca²⁺ release from intracellular stores is necessary but not sufficient to stimulate secretion. Agonist-induced Ca²⁺ release from intracellular stores appears to be necessary to initiate Ca²⁺ influx across the plasma membrane, which in turn provides the sustained elevation in [Ca²⁺]_i required for activation of the receptor-regulated secretory machinery.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Release of Ca2+ from intracellular stores is necessary but not sufficient for BBS-stimulated secretion. Treatment of BON/GRP-R19 cells, in Ca2+-free media containing 1 mM EGTA, with 5 µM thapsigargin (Thaps) induced an increase in [Ca2+]i (A) but not CGA secretion (B), whereas thapsigargin treatment of cells bathed in 2 mM extracellular Ca2+ induced both an increase in [Ca2+]i (A) and peptide secretion (B). Depleting the intracellular Ca2+ stores by pretreatment with thapsigargin blocked subsequent BBS-induced increases in [Ca2+]i (C) and peptide secretion (B). Calcium tracings are the average 340/380 nm ratios recorded from 20-40 individual cells ± S.D. Secretion is expressed as a percentage of plaque-forming cells in 1 h ± S.E. *, p < 0.01 versus 0.5% Me2SO. , p < 0.01 versus thapsigargin (Ca2+free).

BBS-stimulated Secretion Requires PKC and MAPK Activation-- Previous studies have shown that GRP-R is coupled to the activation of both PKC and MAPK/ERK pathways; however, the role of these kinases in GRP-R-regulated secretion is not well defined. Direct activation of PKC with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated secretion of CGA from BON/GRP-R cells (Fig. 7A, 1 µM PMA) without increasing [Ca²⁺]_i (Fig. 7B, 1 µM PMA). PMA also induced MAPK/ERK activation (Fig. 7C, lane 7). The MEK inhibitor PD98059 inhibited PMA-stimulated secretion and MAPK/ERK activation in a dose-dependent manner (Fig. 8, A and B). These data indicate that PMA-sensitive PKC is upstream of MEK and the regulation of peptide secretion in BON/GRP-R cells.

View larger version (38K): [in this window] [in a new window]   Fig. 7.   Effects of a PKC inhibitor, GF109203X (GFX), and a PKC activator, phorbol 12-myristate 13-acetate (PMA) on [Ca2+]i (A), CGA secretion (B), and ERK activation (C). Treatment of BON/GRP-R cells with PMA (1 µM) stimulated secretion in a similar number of cells as BBS (A) and did not stimulate an increase in [Ca2+]i (B). Pretreatment of the cells for 4 min with GFX (5 µM) blocked BBS-stimulated secretion (A) but not agonist-induced increases in [Ca2+]i (B). Secretion data are expressed as a percentage of plaque-forming cells in 1 h ± S.E. Calcium tracings are the average 340/380 nm ratios from 20-40 individual cells ± S.D. *, p < 0.01 versus 0.5% Me2SO (DMSO). C, a Western blot of BON/GRP-R cell proteins was probed for ERK-1 and -2 using an affinity purified rabbit IgG (pTEpY; Promega, Madison, WI) that preferentially detects the dually phosphorylated active form of these enzymes. Cells were pretreated with a MEK inhibitor, PD98059 (50 µM), GFX (5 µM), or PMA (1 µM). The effects of these agents on ERK-1 and -2 activation were assessed ± BBS (106 M) stimulation for 5 min. Control cells were treated with Me2SO at a final concentration of 0.5%.

View larger version (27K): [in this window] [in a new window]   Fig. 8.   The MEK inhibitor, PD98059, blocks PMA-induced secretion (A) and ERK activation (B). A, BON/GRP-R cells were pretreated for 4 min with different concentrations of PD98059 (25, 50, and 100 µM) and then stimulated with PMA (1 µM). The effects of PD98059 on secretion are expressed as a percentage of plaque-forming cells in 1 h ± S.E. *, p < 0.01 versus BBS. B, a Western blot of BON/GRP-R cell proteins probed for activated ERK-1 and -2. DMSO, Me2SO.

To investigate whether PKC was coupled to GRP-R-regulated secretion, cells were pretreated with the PKC inhibitor, GF109203X (GFX) (5 µM), and stimulated with BBS. Pretreatment with GFX completely blocked BBS-stimulated CGA release (Fig. 7A, GFX + BBS) and MAPK/ERK activation (Fig. 7C, lane 6), but did not effect BBS-induced increases in [Ca²⁺]_i (Fig. 7B, 5 µM GFX). These data demonstrate that blocking PKC activation is sufficient to block BBS-induced secretion and MAPK/ERK activation in BON/GRP-R cells even in the presence of an increase in [Ca²⁺]_i, suggesting that one role for BBS-induced increases in [Ca²⁺]_i is to activate Ca²⁺-sensitive PKC isozymes.

MEK Activity Maintains BBS-induced Elevations in [Ca²⁺]_i-- To further investigate the role of MEK-mediated pathways in GRP-R-regulated secretion, BON/GRP-R cells were treated with the MEK inhibitor, PD98059. As expected, PD98059 blocked BBS-induced MAPK/ERK activation (Fig. 7C, lane 5) and caused a dose-dependent inhibition of CGA secretion (Fig. 9A). However, in contrast to the PKC inhibitor (GFX), which did not affect agonist-induced changes in [Ca²⁺]_i, PD98059 induced a dose-dependent inhibition of BBS-stimulated increases in [Ca²⁺]_i (Fig. 9B). To evaluate the specificity of the PD98059 effect, BON/GRP-R cells were transfected with either a vector construct containing a dominant negative mutant form of MEK-1 (MEK-2A-EECMV; Dr. Dennis J. Templeton, Case Western Reserve University) or the empty expression vector EECMV. Like PD98059, the dominant negative MEK-1 blocked BBS-stimulated increases in [Ca²⁺]_i (Fig. 9C, dnMEK-1). Transfection of the empty expression vector had no effect on BBS-induced [Ca²⁺]_i responses (Fig. 9C, Vector). Together, these data demonstrate a novel role for MEK-mediated pathways in the maintenance of agonist-induced increases in [Ca²⁺]_i and suggest that MEK modulates GRP-R-regulated exocytosis, in part, by affecting the activity of Ca²⁺-sensitive PKC.

View larger version (32K): [in this window] [in a new window]   Fig. 9.   Inhibition of MEK blocks BBS-stimulated CGA secretion (A) and agonist-induced increases in [Ca2+]i (B and C). A, BON/GRP-R19 cells were pretreated for 4 min with different concentrations of PD98059 (1, 5, and 10 µM) and then stimulated with BBS (107 M). The effects of PD98059 on secretion are expressed as a percentage of plaque-forming cells in 1 h ± S.E. from three separate experiments. B, calcium tracings are the average 340/380 ratios from 20-40 individual cells ± S.D. *, p < 0.01 versus BBS. C, cells were cotransfected with either a dominant negative mutant form of MEK-1 (dnMEK) or the empty expression vector (Vector) and an expression containing a cDNA for green fluorescence protein (GFP). After 24 h, the cells were loaded with fura-2 and cells expressing green fluorescence protein were identified. Then, the cells were stimulated with BBS (107 M) and changes in [Ca2+]i were recorded. Like PD98059, dominant negative MEK inhibited BBS-stimulated increases in [Ca2+]i. Transfection of the empty vector and green fluorescence protein did not affect BBS-stimulated [Ca2+]i responses. DMSO, Me2SO.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

An important trigger of receptor-regulated secretion is an agonist-induced increase in [Ca²⁺]_i (28-32). The development of techniques that allowed changes in [Ca²⁺]_i to be studied within individual cells has revealed complex patterns in calcium signals, including repetitive oscillations, in response to physiological concentrations of agonist (11, 33-35). In this study, we have shown that stimulation of BON/GRP-R cells with low concentrations of BBS induces oscillatory changes in [Ca²⁺]_i, whereas high concentrations of BBS produce a biphasic calcium response that lasts up to 20 min in normal extracellular calcium. BBS-induced calcium oscillations appear to be a general characteristic of GRP-R activation. We have investigated GRP-R-mediated [Ca²⁺]_i signaling in several cell lines, including a human gastric carcinoma cell line (SIIA) and a human prostate cancer cell line (PC3), both of which express native GRP-R, as well as another transfected cell model, GRP-R-transfected mouse NIH Balb/C 3T3 fibroblasts. In these cell lines, low concentrations of BBS stimulate [Ca²⁺]_i oscillations. BBS-induced calcium oscillations also have been reported in the insulin-secreting cell line, HIT-T15 (11), and in the pancreatic acinar cell line, AR4-2J (34). However, this is the first report to address the role of BBS-induced [Ca²⁺]_i oscillations in GRP-R-regulated exocytosis.

A central question in calcium signaling biology is whether [Ca²⁺]_i oscillations specify receptor- or cell type-specific information. Because of the intimate association of receptor-induced calcium signals and secretion, [Ca²⁺]_i oscillations may represent a coded signal that modulates the exocytotic machinery. Recently, experimental evidence obtained from isolated pituitary cells has supported this hypothesis. Tse and co-workers (28) have shown that stimulation of gonadotrophs with gonadotropin-releasing hormone induces oscillations in [Ca²⁺]_i that are temporally associated with an increase in cell membrane capacitance, a measure of vesicle fusion with the plasma membrane. Additionally, constitutive secretion of growth hormone, from somatotropes, increases with both elevations in the frequency and amplitude of spontaneous [Ca²⁺]_i oscillations (31).

In contrast to pituitary cells, the data presented here show that BBS-induced peptide secretion from BON/GRP-R cells was associated with a biphasic, sustained elevation in [Ca²⁺]_i and not [Ca²⁺]_i oscillations. It is not clear why [Ca²⁺]_i oscillations are not associated with secretion from these cells; however, our results are consistent with recent reports showing that GRP-R-regulated secretion is associated with a sustained elevation in [Ca²⁺]_i in two other cell types: primary cultures of canine G-cells (30) and the mouse intestinal cell line, STC-1 (32). Seensalu and co-workers (30) showed that BBS-stimulated gastrin secretion from G-cells was inhibited when the second phase of a biphasic [Ca²⁺]_i response was blocked by removal of extracellular Ca²⁺. Similarly, a sustained biphasic [Ca²⁺]_i response, dependent on the presence of Ca²⁺ in the extracellular solution, correlated with BBS-stimulated cholecystokinin release from STC-1 cells (32). The fact that BBS-induced secretion from BON/GRP-R requires a sustained elevation in [Ca²⁺]_i like these other cell types suggests a common mechanism for GRP-R-regulated exocytosis and demonstrates the utility of the BON/GRP-R cell line has a model for studying BBS-induced secretion.

Bombesin-induced secretion requires an increase in [Ca²⁺]_i that is initiated by release of Ca²⁺ from intercellular stores and sustained by an influx of Ca²⁺ across the plasma membrane. Calcium influx into excitable cells can occur through either voltage-gated Ca²⁺ channels or by various transport mechanisms. In non-excitable cells, influx of extracellular Ca²⁺ occurs by either capacitative Ca²⁺ uptake through non-voltage-gated, store-operated Ca²⁺ channels activated by depletion of intracellular Ca²⁺ pools (36, 37); through nonspecific receptor- or second messenger-operated cation channels (38); or by sodium-calcium exchange (39). In this study, current-voltage analysis using whole cell patch voltage clamp showed that there are voltage-gate calcium currents on BON/GRP-R cells. However, the cells were not depolarized by BBS and inhibitors of voltage-gated channels did not block BBS-stimulated increases in [Ca²⁺]_i. It is unknown whether the predominant mechanism for calcium influx into BON/GRP-R cells is through calcium release-activated channels or nonspecific receptor or second messenger-operated cation channels. However, it is clear from the data presented that Ca²⁺ release from intracellular stores is necessary to stimulate Ca²⁺ influx across the plasma membrane in BON/GRP-R cells. Blocking PLC- activation with U73122 or depleting intracellular Ca²⁺ stores by pretreating with thapsigargin completely blocked BBS-induced increases in [Ca²⁺]_i.

An increase in [Ca²⁺]_i will not stimulate secretion in the absence of an activation of both PKC- and MEK-regulated pathways. Three groups of PKC isozymes have been identified, based on biochemical properties and sequence homologies. These include the conventional, novel, and atypical kinases. The conventional PKC group, which includes PKC-, -₁, -₂, and -, are activated by phorbol esters, DAG, and Ca²⁺. The novel PKC isozymes are activated by phorbol esters and DAG but not by Ca²⁺. The activity of the atypical kinases are independent of phorbol esters, DAG, and Ca²⁺. The observation that PMA is sufficient to stimulate CGA secretion in the absence of an increase in [Ca²⁺]_i indicates that the basic secretory machinery in BON/GRP-R cells can be regulated by PKC activation alone and suggests the possible involvement of both the conventional and novel PKC isozymes. However, the dependence of GRP-R-regulated secretion on a rise in [Ca²⁺]_i suggests that conventional PKC isozymes are the most likely mediators of the BBS-induced response in these cells.

Recently, several downstream targets of PKC phosphorylation have been identified that may be important in the process of receptor-regulated exocytosis. Several neuron-specific proteins that function in vesicle docking and fusion at presynaptic membranes have been shown by in vitro kinase assays to be substrates for PKC phosphorylation, including the proteins, syntaxin-1 and -4 (40), which are components of the soluble N-ethylmaleimide-sensitive attachment factor receptor complex. Additionally, the myristoylated alanine-rich protein kinase C substrate (MARCKS) protein is a target of PKC. MARCKS proteins have been implicated in neurosecretion and have been shown to be phosphorylated by PKC in synaptosome preparations. Liu and co-workers (41) have demonstrated a close temporal association between arginine vasopressin-induced MARCKS phosphorylation and secretion of adrenocorticotropin from ovine anterior pituitary cells, suggesting that MARCKS may be involved in the initial PKC-dependent intracellular events underlying exocytosis of this hormone. Non-neuronal cells express isoforms of these various proteins; however, it remains to be determined which, if any, play a role in GRP-R-regulated exocytosis in BON/GRP-R cells.

Previous studies have demonstrated a link between MAPK/ERK- and PKC-regulated pathways in receptor-mediated secretion from various cells types (14, 42-44). We have found that GRP-R-regulated secretion in BON/GRP-R cells also involves the activation of both PKC and MAPK/ERK pathways. Similar to PKC, MAPK/ERK have been shown to phosphorylate synaptic vesicle proteins involved in exocytosis such as synapsin I (45). In addition to their potential role in the regulation of proteins involved with vesicle fusion, we have found that MAPK pathways can regulate secretion by affecting agonist-induced changes in [Ca²⁺]_i.

Three related MAPK cascades have been identified in mammalian cells and are named according to the final enzyme in each pathway. They are the extracellular signal-regulated protein kinase (ERK) pathway, the c-Jun N-terminal kinase pathway, and the p38 MAP kinase pathway. Recently, Malgorzata and co-workers (46) have shown that a member of the p38 MAP kinase cascade can regulate [Ca²⁺]_i. They reported that p38-2 is selectively activated by bradykinin in NG108-15, cells leading to a slow inhibition of an N-type Ca²⁺ current. We show that blocking MEK with either PD98059 or a dominant negative mutant of MEK-1 inhibits BBS-induced increases in [Ca²⁺]_i, suggesting that the role of active MEK is to maintain elevated [Ca²⁺]_i during agonist stimulation. MEK-1 is an upstream dual-specificity kinase that phosphorylates both threonine and tyrosine residues on ERK-1 and -2. Expression of a constitutively active mutant form of MEK will activate p38 (47). However, it is unlikely that the effects of PD98059 or dominant negative MEK on BBS-stimulated increases in [Ca²⁺]_i are due to regulation of p38 in BON/GRP-R cells. Inhibition of MEK blocks BBS-induced increases in [Ca²⁺]_i in BON/GRP-R cells, whereas p38-2 activation by bradykinin in NG108-15 cells has the opposite effect on [Ca²⁺]_i by inhibiting an inward N-type Ca²⁺ current. It is not known whether MEK regulation of [Ca²⁺]_i in BON/GRP-R cells is mediated by either ERK-1 and/or -2 or whether an undefined pathway is involved. However, the data presented in this study, together with the previous work of Malgorzata and co-workers, suggest that multiple MAP kinase cascades are involved in receptor regulation of [Ca²⁺]_i, presenting the possibility that it is a general characteristic of this important family of kinases.

In conclusion, we have developed a model human cell line to investigate the molecular mechanisms of GRP-R-regulated exocytosis. Fig. 10 summarizes our proposed model for GRP-R-mediated secretion. We have found that peptide secretion involves activation of PLC leading to an increase in [Ca²⁺]_i that is initiated by Ca²⁺ release from intracellular stores and sustained by influx across the plasma membrane. Agonist-induced increases in [Ca²⁺]_i, however, will not stimulate peptide release in the absence of PKC and MEK activation, suggesting that a role of Ca²⁺ is to activate conventional PKC isozymes, which, in turn, activate ERK through MEK. The role of MEK-mediated pathways in GRP-R-regulated exocytosis are to, in part, maintain elevated levels of [Ca²⁺]_i, and perhaps the activity of Ca²⁺-sensitive PKC isozymes through a feedback loop mechanism. Future studies will attempt to identify the mechanism by which MAPK regulates agonist-induced increases in [Ca²⁺]_i.

View larger version (19K): [in this window] [in a new window]   Fig. 10.   Model of GRP-R-regulated secretion in BON/GRP-R cells.

	ACKNOWLEDGEMENTS

We thank Dr. Alan P. Fields for critical review of the manuscript and helpful comments and Dr. Dennis J. Templeton, Department of Pathology, Case Western Reserve University, for providing the dominant negative mutant of MEK1 (MEK-2A-EECMV) and control vector (EECMV). We also thank Eileen Figueroa, Liz Cook, Karen Martin, and Steve Schuenke for assistance in the preparation of this manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant P01 DK35608.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Dept. of Surgery, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0722. Tel.: 409-772-1845; Fax: 409-772-6368; E-mail: mhell mic{at}utmb.edu.

	ABBREVIATIONS

The abbreviations used are: BBS, bombesin; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; CGA, chromogranin A; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; G-418, Geneticin; GRP, gastrin-releasing peptide; GRP-R, gastrin-releasing peptide receptor; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK kinase; NT, neurotensin; RHPA, reverse hemolytic plaque assay; GFX, GF109203X; PKC, protein kinase C; RIA, radioimmunoassay; KRH, Krebs-Ringer-Hepes; PLC, phospholipase C; DAG, diacylglycerol; TBS, Tris-buffered saline; MARCKS, myristoylated alanine-rich protein kinase C substrate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES