Characterization of the release of cholecystokinin from a murine neuroendocrine tumor cell line, STC-1

ELSEVIER

Biochimica et Biophysica Acta 1221 (1994) 339-347

Biochi:ic~a et BiophysicaA~ta

Characterization of the release of cholecystokinin from a murine neuroendocrine tumor cell line, STC-1 Cecilia H. Chang

a, W i l l i a m Y . C h e y a, Q i S u n

a A n d r e w L e i t e r b T a - m i n C h a n g a,,

a Centerfor Digestive and Liver Diseases, Department of Medicine, Universityof Rochester Medical Center, Box Medicine, 601 Elmwood Avenue, Rochester, NY 14642, USA b Tufts University School of Medicine, Department of Medicine, Boston, MA, USA (Received 1 September 1992; revised manuscript received 4 August 1993)

Abstract

The murine neuroendocrine cell line, STC-1, was found to contain 296.8 + 1.8 fmol of cholecystokinin-like immunoreactivity (CCK-LI) per mg cell protein. Immunocytochemieal stain of STC-1 cells maintained in monolayer culture indicated that CCK-LI activity was present in 93% of the cells. Analysis by reverse-phase high-performance liquid chromatography indicated that STC-1 cells contained and an unidentified form as the predominant storage form. However, only CCK-8 was released into the culture medium upon stimulation by various secretagogues. The release of CCK-LI from STC-1 cells was stimulated by dibutyryl cAMP, forskolin, KCI, A23187, 4/3-phorbol 12-myristate 13-acetate and luminal stimulants, e.g., sodium oleate, L-tryptophan, camostat and plaunotol. The release of CCK-LI from STC-1 cells was also stimulated by a neuropeptide, bombesin. The stimulatory effects of most of these agents were dose dependent. The stimulatory effects of dibutyryl cAMP, forskolin, and plaunotol were potentiated by 3-isobutyl-l-methyl xanthine, while that of camostat was not. The results obtained in this study indicate that the release of CCK from STC-1 cells shares the same characteristics of CCK release as from the CCK-secreting cells of the intestinal mucosa observed both in the dog and the rat in vitro and in vivo. Thus, the cellular mechanism of CCK release which appears to be cAMP- and Ca2+-dependent may be modulated by cellular protein kinase C activity. The STC-1 cell appears to be a suitable model for studying the mechanism of CCK release.

CCK-8

Keywords:Cholecystokinin; Protein kinase C; cyclic AMP; Calcium ion; Bombesin 1. Introduction

Cholecystokinin (CCK) is an important gastrointestinal hormone which stimulates pancreatic exocrine secretion and gallbladder contraction, and inhibits gastric emptying and acid secretion [1,2]. In addition to its presence in the mucosal endocrine cells of the small

* Corresponding author. Fax: + 1 (716) 2717868. Abbreviations: BSA, bovine serum albumin; camostat, commercial name of N-dimethylcarbamoyl 4-(4-guanidinobenzoyloxy) phenylmethane suifonate; CCK, cholecystokinin; CCK-8, CCK-33 .... and CCK-n, cholecystokinin peptides of 8, 33, or n amino-acid residues; CCK-LI, CCK-like immunoreactivity; DBcAMP, dibutyryl cyclic adenosine-3',5'-monophosphate; DMEM, Dulbecco's modified essential medium; DPBS, Dulbecco's phosphate-buffered saline; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HPLC, highperformance liquid chromatography; IBMX, 3-isobutyl-l-methylxanthine; 4a-PMA, 4a-phorbol 12-myristate 13-acetate; 4fl-PMA, 4/3-phorbol 12-myristate 13-acetate. 0167-4889/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 8 8 9 ( 9 3 ) E 0 2 0 9 - I

intestine, CCK is also present in both the central and peripheral nervous system and acts as a neuromodulator [3]. This hormone exists in multiple molecular forms, including CCK-58, CCK-39, CCK-33, CCK-22, CCK-12 and CCK-8 [2,3]. The presence and concentration of these molecular forms appear to vary with the tissue and species studied, due to variations in post-translational processing of the preprohorrnone [2,3]. In the intact animals, CCK is released by ingestion of a mixed meal [4-6], amino acids [7], fat [8,9] and a synthetic serine protease inhibitor, camostat [10]. The release of CCK is also stimulated by intraduodenal infusion of protein [11], amino acids [5,12], fats [13,14] and sodium oleate [15]. Electrical stimulation of the vagus nerve [16,17] and intravenous infusion of gastrin-releasing peptide [18] or its analog bombesin [19] also elicited the release of CCK. However, the mechanisms through which these stimulants effect CCK release are not clear.

340

C.H. Chang et al./ Biochimica et Biophysica Acta 1221 (1994) 339-347

Studies with partially purified CCK-containing cells isolated from the mucosae of canine jejunum [20,21] and rat duodenum [22] have indicated that cellular release of CCK involves both cAMP-[20-22] and Ca 2+dependent mechanisms [21]. The release of CCK from the rat and canine cells is also stimulated by fl-PMA [21,22], a potent stimulator of protein kinase C. However, due to heterogeneity of these cell preparations, it is not known from these studies whether or not all these regulatory mechanisms operate in the CCK-releasing endocrine cells. Recently, a CCK-secreting tumor cell line [23] and hybridoma cells derived from rat intestinal mucosa [24] have been established. We have found that the secretin-secreting cell line, STC-1 (which was derived from an intestinal tumor produced in transgenic mice carrying insulin promotor and a viral oncogene [25]) also expresses and secretes CCK. Due to their high degree of homogeneity, these cells may be good models for studying the mechanism of gastrointestinal hormone release, provided that they are stimulated by the same agents capable of mediating the release of these gastrointestinal hormones. The purpose of the present study is to characterize the response of STC-1 cells to various stimulating agents capable of releasing CCK from the mucosal cell preparations [20-22]. The results presented in the present report indicate that STC-1 cells are stimulated to release CCK by the same luminal stimulants or secretagogues as those described for the intestinal mucosal preparations. In addition, we have found that the release of CCK from these cells is also stimulated by bombesin and plaunotol, an anti-ulcer agent reported to stimulate pancreatic exocrine secretion of both bicarbonate and protein [26] and that the effect of plaunotol is probably mediated through a cAMP-dependent mechanism.

2. Materials and methods

Materials Forskolin, dibutyryl cyclic adenosine monophosphate (DBcAMP), A23187, 4/3-phorbol 12-myristate 13-acetate (/3-PMA), /3-NADH, and gentamycin sulfate were obtained from Sigma, St. Louis, MO. 4aPMA was obtained from LC Services, Worburn, MA. Oleic acid was obtained from Fisher Scientific, Fairlawn, NJ. Percoll was obtained from Pharmaeia LKB Biotechnology, Piscataway, NJ. Camostat {N,N-dimethylearbamoyl-4-(4-guanidinobenzoyloxy)phenylacetate methanesulfonate} was a gift of Ono Pharmaceuticals, Tokyo, Japan. Lactate dehydrogenase was obtained from Worthington Biochemical, Freehold, New Jersey. Bombesin was purchased from Peninsula Laboratories, Belmont, CA. Plaunotol (7-hydroxylmethyl3,11,15-trimethyl-2,6,10,14-hexadecatetranen-l-ol) was

a gift from Sankyo Pharmaceuticals, Tokyo, Japan, through arrangement by Dr. K. Shiratori of Tokyo Women's Medical College. Dulbecco's modified essential medium, horse and fetal bovine sera and a mixture of penicillin and streptomycin and Earle's balance salt were purchased from Grand Island Biological, Grand Island, NY. Tissue culture plates (6-welled) were obtained from Costar, Cambridge, MA. STC-1 cells were obtained from Dr. Seth Grant, Columbia University, NY. The immunohistochemical stain kit (streptoavidin-biotin kit for rabbit primary antibody) was obtained from Zymed Laboratories, South San Francisco, CA.

Preparation of the STC-1 cells The STC-1 cells were maintained in DMEM containing 15% horse serum and 2.5% fetal bovine serum, streptomycin (100/zg/ml), penicillin (100/zU/ml) and gentamycin sulfate (50/zg/ml) in a humidified incubator at 37°C under 5% CO2/95% air. For studying the release of CCK, the cells were seeded into 6-welled culture plates at 1 • 105 cells/ml and cultured for 4 to 5 days until 80% confluency. The cell number, as determined after resuspension with trypsin treatment, reached 2.106 cells/well.

Studies on the release of CCK On the day of the experiment, the medium was removed from the cultures of STC-1 cells, and the cells were washed once with Earle's balanced salt solution containing 10 mM Hepes (pH 7.4), 5 mM sodium pyruvate, 0.01% soybean trypsin inhibitor and 0.2% BSA. The cells were then incubated in 0.5 ml of the same buffer in the absence or presence of various stimulants at 4°C or 37°C for 120 min or for the periods of time as specified. The incubation was stopped by cooling the cell culture plates on ice. The medium was removed for radioimmunoassay of CCK. The cell was extracted in 1 ml of 0.1 M HC1 and lyophilized before being reconstituted and assayed for CCK. The amount of CCK-LI released into the medium was expressed as a percentage of the total CCK-LI cell content (medium plus cell in each well). All the data are represented as the mean +_ S.E. averaged from the mean of duplicate wells of several experiments.

High-performance liquid chromatography (HPLC) The molecular forms of CCK-like immunoreactivity (CCK-LI) released into the medium and those stored in STC-1 cells were determined by HPLC after extraction with C-18 Sep-Pak cartridges using the same chromatographic conditions as described in a previous report [27].

Radioimmunoassay of CCK and immunocytochemical stain of STC-1 cells Radioimmunoassay of CCK was carried out as described [27]. Immunocytochemical stain of STC-1 cells

C.H. Chang et al. / Biochimica et Biophysica Acta 1221 (1994) 339-347

was carried out with cells grown on a glass cover slip for 3 or 4 days. T h e cells were washed at 4°C in Dulbecco's phosphate-buffered saline and then fixed in Bouin's solution for 15 rain at room t e m p e r a t u r e [22]. The fixed cells were incubated with 0.15% Triton X-100 in DPBS for 30 min at room temperature, washed

three times with DPBS and then incubated in 3% in DPBS for 30 min. The cover slip was then incubated with either a normal rabbit serum or the rabbit anti-CCK serum used for radioimmunoassay (both at 1:12800 in DPBS containing 0.1% BSA) at 4°C for 24 h. The cover slip was washed and then H202

IP

A

;~

341

~

~

~

-

....

~ 4

~i~ i~ii~i ¸ . . . . .

Fig. 1. Immunohistochemical stain of CCK-containing cells in STC-1 cells. (A) Cells stained with an anti-CCK serum (1 : 12 800); (B) cells stained with normal rabbit serum (1:12 800). Magnification: x 400.

342

C.H. Chang et al. / Biochimica et Biophysica Acta 1221 (1994) 339-347

incubated with a biotinylated goat anti-rabbit immunoglobin followed by streptoavidin-horseradish peroxidase conjugate according to the procedure described in the staining kit provided by the manufacturer. The CCK cells were visualized after adding diaminobenzoic acid substrate solution followed by a wash with DPBS. Pictures were taken from fields randomly selected from five slides. CCK-containing cells were then counted from the prints seperately by two of the authors.

Statistical analysis Statistical analysis of the results was performed by analysis of variance for experiments with a single factor for comparison of multiple means using the post hoc procedure of honestly significant difference of Tukey as described by Winer [28]. All the calculations were performed on a Gateway 2000 (386SX/25 MHz) personal computer, using the Systat software program (Systat, Evanston, IL). For the time-course studies, the CCK-LI release stimulated by each agent was compared with the corresponding control at each time point using Student's paired t-test. Differences among means with P < 0.05 were regarded as significant.

inhibited (o). At 37°C, and in the absence of a secretagogue, there was a small basal secretion of CCK-LI (3.5% per h) that persisted for 2 h (o). At the same temperature and in the presence of 1 mM DBeAMP plus 0.5 mM IBMX (4), the release of CCK was stimulated for 30 min and then leveled off. Addition of forskolin (30/xM) in the presence of the same concentration of IBMX (/x ) resulted in a continuous stimulation of CCK-LI release over the entire period of incubation. In the presence of 0.1 ~M 4/3-PMA, the release of CCK-LI was linearly stimulated for 60 min and then leveled off ([]). Although the kinetics of the release of CCK varied with these and other stimulants, the amount of CCK released after 2 h of incubation was invariably greater than that of control. Therefore, in subsequent studies, the cells were incubated with each stimulant for 2 h. It should be mentioned that under all the conditions, none of the stimulants at the highest concentration we used in the present study caused an increased release of lactate dehydrogenase, or inclusion of trypan blue (data not shown), which indicated that the large quantity of CCK-LI released in the presence of a high concentration of forskolin, 4/3PMA or camostat (see below) was not due to cell breakage.

3. Results

CCK-LI in STC-1 cells The concentration of CCK-LI in the STC-1 cells was 296.8 + 1.8 f m o l / m g cell protein. Immunocytochemical stain of STC-1 cells demonstrated that 93.0 + 0.6% (n = 5) of the cells contained CCK-LI (Fig. 1). Reverse-phase HPLC of the cell extract, as shown in Fig. 2A, indicated that there were two major forms of the CCK-LI stored in STC-1 cells which had retention times corresponding to those of sulfated CCK-8 (32 min, 20.7%) and an unidentified form of CCK-LI (41 min, 54.9%). The oxidized form of CCK-8 eluted at 12 min might be derived from CCK-8 during processing of the cell extract. Thus, the precursors of CCK were probably processed post-translationally in STC-1 cells to the small molecular forms. As shown in Fig. 2B, analysis of the CCK-LI released into the medium when stimulated by a synthetic serine protease inhibitor, camostat, indicated that only CCK-8 was released from the STC-1 cells. A similar chromatogram was obtained from the medium collected after stimulation with 1 /xM of 4/3-PMA (data not shown). Time-course of CCK release We studied the effect of various secretagogues on STC-1 cells which are known to stimulate the release of CCK from isolated mucosal cells [20-22] or in vivo. As shown in Fig. 3, the cells released very little CCK-LI over a period of 2 h at 4°C when exocytosis was

CCK-B(ns) pCCK-33

500

A

40O

CCK-B(ox) $

30O

CCK-8

cCCK-58

/-

6om [...

40 N

200

o

o 20 N

6O0

I cD CD

B

5OO

60 m

400 3O0 2OO

20 N

100 , 0

~

~ .... 0 -40 60 80 0 20 ELUTION TIME (min)

Fig. 2. Analysis of molecular form of C C K in STC-I cells. Cell extract (A) and the incubation medium (B) of STC-1 cells, after Sep-Pak extraction, were subjected to H P L C on a C-18 column as described in Section 2. The medium after stimulation with 2 m M camostat was analyzed in B. The arrows denote the rctention times of some standard peptides chromatographed under the same conditions.

C.H. Chang et aL / Biochimica et Biophysica Acta 1221 (1994) 339-347

343

.,J

40

o

o

cJ

"

3O cj

,

J, 5

5')

<

,.-1

10

(3 r~ rj

0

I

~ 0

30

60

90

I

0

0

120

/t

I

I

I

0.1

I

1

A23187 (/zM)

INCUBATION TIME ( m i n ) Fig, 3. Time-courses of the release of CCK from STC-1 cells after stimulation with secretagogues. STC-1 cells were incubated at 4°C or 37°C in the absence or presence of various stimulants and incubated for the periods of time indicated. The amount of CCK-LI released into the medium was determined by radioimmunoassay. The data represent the means±S.E, from three experiments. The symbols represent the following conditions: ( o ) control at 4°C; (e) control at 37°C; (zx) 30/zM forskolin plus 0.5 mM IBMX; ( A ) 1 mM DBcAMP plus 0.5 mM IBMX; (D) 0.1 /zM 4/3-PMA; * indicates a significant increase in CCK-LI release over the control ( P < 0.05) at the corresponding time after correction using 4°C control.

The effect of various agents on the release of CCK-LI The results as summarized in Table 1 indicate that the release of CCK-LI from STC-1 cells was stimulated significantly by DBcAMP, forskolin, 4/3-PMA, A23187, KC1, L-tryptophan, sodium oleate, camostat, and bombesin at the concentrations tested. IBMX by itself did not stimulate the release of CCK-LI at concentrations below 0.5 mM.

Fig. 4. Dose-dependent effect of the calcium ionophore, A23187, on CCK-LI release from STC-1 cells. All data were corrected using the control at 4°C. The data represent the averages from five experiments. The symbols * and * * depict P < 0.05 and P < 0.01 vs. the control, respectively.

Dose-dependent effects of various secretagogues The stimulatory effects of some of the above secretagogues were also dose dependent. Addition of A23187 from U.05 to 1.0/.~M resulted in a progressive increase in CCK-LI release that was statistically significant at the dose of 0.1 /xM or greater (Fig. 4). In the concentration range of 0.01 to 1/~M, 4/3-PMA also stimulated the release of CCK-LI in a dose-dependent manner (Fig. 5). On the other hand, 4a-PMA, had no effect on CCK release (data not shown). Addition of bombesin from 0.1 pM to 10 nM also progressively increased the release of CCK-LI, reaching statistical significance at 1 nM and 10 nM (Fig. 6). Incubation of STC-1 cells with DBcAMP from 0.1 to 2 mM resulted in a small increase in CCK-LI release, reaching statistical signifi-

Table 1 The effects of secretagogues on the release of CCK-LI from STC-1 cells Agent

A23187 /3-PMA KC1 IBMX DBcAMP Forskolin Bombesin Sodium oleate L-Trp Camostat Plaunotol

Concentration

1.0/zM 0.1 ~tM 50.0mM 0.5 mM 2.0mM 30.0/xM 0.1 nM 0.2 mM 20.0 mM 1.0 mM 0.1 mM

n

5 9 3 4 4 4 5 9 3 4 4

CK-L1 Release (% of cell content) Control

+ Agent

3.6+0.5 3.4±0.5 3,3+0.5 4,2±0.6 4.2±0.6 4,2±0.6 5,1±0.9 5.5 ± 0.7 5.5 ± 0.8 5.0±0.9 4.75:1.2

7.6±1.0 31.~1±0.9 35.4±2.7 5.15:0.8 6.6±0.7 13.8±1.9 8.9±0.8 10.9 ± 1.0 9.9 ± 1.6 10.1± 1.7 9.3±1.2

Stimulation (%)

~- .50 ¢D tO

o

112±28 ** 826±26 ** 989±82"* 21+20 58±17 * 231±46 ** 745:15 * 97 ± 19 * 79 ± 29 * 116-t-34 ** 98±24 *

The above data represent m e a n s + S.E. of n experiments. * and ** denote P < 0.05 and P < 0.01, respectively, when pared with the control.

com

40

N 30 <

20

-d

Q:

10

..J

I

o

olol

o'.1

4fl-PMA (/.~M) -

Fig. 5. Dose-dependent effect of 4/3-PMA on CCK release from STC-1 cells. All data were corrected as described in the legend to Fig. 4 (n = 6; * *, P < 0.01 vs. control).

C.H. Chang et al. / Biochimica et Biophysica Acta 1221 (1994) 339-347

344

A ,#t

40 Q)

T CA

o o

AA 30

o

20 r~ M

10

I

M

I

0

-11

-i0

-9

LOG BOMBESIN

v

-8

cance only at the highest concentration (Fig. 7, open symbols). In the presence of 0.5 mM IBMX, the effect of DBcAMP was enhanced at all doses and became statistically significant at a dose of 0.1 mM and reached a maximal effect at 0.5 mM (Fig. 7, filled symbols). However, there was no clear dose-response relationship in either case. Forskolin in doses ranging from 1 to 30 ~M resulted in a dose-dependent stimulation of CCK-LI release from STC-1 ceils (Fig. 8, open symbols). The effect of forskolin was substantially enhanced by 0.5 mM IBMX so that at 0.1 /zM it already had a significant stimulatory effect on CCK-LI secretion (Fig. 8, filled symbols). Addition of L-tryptophan

** CA

/

~-II .

0.0

.

.

.

.

0.1

1.0

FORSKOLIN

Fig. 6. Dose-dependent effect of bombesin on CCK release from STC-1 cells. All data were corrected as described in the legend to Fig. 4 (n = 5; *, P < 0.05 vs. control).

O c9

0

~.)

CONC. (M)

~

10.0

(/~M)

Fig. 8. Dose-dependent effect of forskolin on CCK release from STC-1 cells. The data represent the means:t: S.E. from four experiments after correction using 4°C controls. ( o ) In the absence of IBMX; (e) in the presence of 0.5 mM IBMX. The asterisks denote a statistical significance vs. the corresponding control, whereas the triangles denote a statistical significance between the two experimental conditions ( + IBMX), as described for Fig. 7.

from 5 to 20 mM and sodium oleate from 50 to 200 /~M also gradually increased CCK-LI release but their effects were significant only at the highest concentration tested (data not shown). As shown in Fig. 9, addition of plaunotol from 10 to 100/zM resulted in a small increase in CCK release from STC-1 cells, albeit reaching statistical significance only at the highest concentration tested (open symbols). Interestingly, the effect of plaunotol was enhanced 2-fold in the presence of IBMX, leading to a linear dose-dependent release of CCK-LI (filled symbols). In the presence of increasing concentrations of camostat (0.5 to 4 mM), there

A o

10

o o 03 ,<

20

AA

o (9

,..) v

I

Y

r~a r/)

0

L

0.0

0.5

i

i

1.0 1.5 DBeAMP (rnM)

i

10

M

2.0

Fig. 7. Effect of DBcAMP, in the absence or presence of IBMX, on CCK release from STC-1 cells. ( o ) Without IBMX; (e) with I B M X (0.5 mM). * and * * are P < 0.05 and 'P < 0.01 vs. the corresponding controls, respectively. The triangles denote statistical significance between the points with and without I B M X at the same D B c A M P concentration; ( zx ) P < 0.05; ( ,x zx ) P < 0.01. The data represent the average of the results of four experiments after correction using 4°C control.

r~

I

0

~

L

i

J

0

i0

50

I00

PLAUNOTOL

(/zM)

Fig. 9. Dose-dependent effect of plaunotol on C C K release from STC-1 cells. ( o ) Without IBMX; (e) with I B M X (n = 4). The asterisks and triangles denote statistical significance as described for Fig. 7.

C.H. Chang et al. / Biochimica et Biophysica Acta 1221 (1994) 339-347

20

Q

i0 bQ < r~ r~

i

0

i

I

i

I

0

I

2

3

CAMOSTAT

(mM)

4

Fig. 10. Concentration-dependent effect of camostat on CCK release from STC-1 cells. The asterisks denote P < 0.05 (*), or P <0.01 ( * * ) vs. control.

was a progressive increase in CCK-LI release (Fig. 10) that was not significantly affected by the presence of IBMX (data not shown).

4. Discussion

The results of the present study have indicated that the murine neuroendocrine tumor cells, STC-1, express and secrete CCK-LI. The predominant forms of CCKLI stored in these cells appear to be sulfated CCK-8 and an unidentified form which was eluted at 41 min (slightly behind un-sulfated CCK-8, 38 min) as determined by reverse-phase HPLC. A similar pattern was observed using extracts of rat duodenal mucosa (Chang and Chey, unpublished data). Both sulfated CCK-8 and unsulfated CCK-8 were also found in rat medullary thyroid carcinoma cells [29]. Thus, it is likely that in rodents, CCK is processed post-translationally into small molecular forms. Upon stimulation with a stimulant such as camostat, STC-1 cells released sulfated CCK-8 into the medium, but the unidentified form was not secreted. This pattern of CCK release was also observed when a stronger stimulant, 4/3-PMA (1 tzM) was used. Thus, it was unlikely that the selective release of CCK-8 was due to selective release of different form of CCK in response to different stimulants. It should be noted that prohormone-processing endopeptidases are known to exist in secretory granules [30,31], and that the C-terminal a-amidation monooxygenase is widely distributed in various cell types, including both endocrine and non-endocrine cells [32-34]. Thus, variation in the activities of these enzymes in different tissues or cell types could lead to differences in the post-translational processing of a prohormone such as pro-opiomelanocortin [31] or pro-somatostatin [35]. Variation in sulfation by protein tyrosyl sulfotransferase activity in the trans-Golgi membranes [36,37]

345

could account for the well-documented partial sulfation of fl-lipotropin [37,38], gastrin [39] and CCK-8 in rat medullary thyroid carcinoma cells [29]. Rat medullary thyroid carcinoma cells also secrete CCK-33, glycine-extended CCK-8 and proCCK in addition to the two forms of CCK-8 [29]. Therefore, it cannot be ruled out at present that STC-1 ceils also store and secrete C-terminally extended forms of CCK which might not be detected by our radioimmunoassay. Alternatively, the CCK-LI with the retention time of 41 min might have been the glycine-extended form of sulfated CCK-8 rather than a higher molecular weight form of CCK such as CCK-12 or CCK-22. Due to the lack of a standard of glycine-extended sulfated CCK-8, CCK-12 or CCK-22, this question can not be answered at present. The release of CCK from STC-1 cells was stimulated by many secretagogues and luminal stimulants known to elicit CCK release both in vivo and in vitro. These included four categories of agents: (i) cAMP analog and activators of adenylate cyclase such as DBcAMP and forskolin, respectively; (ii) agents affecting ion transport such as KCI and A23187; (iii) activators of protein kinase C such as 4/3-PMA; and (iv) the luminal stimulants such as L-tryptophan, sodium oleate, camostat and plaunotol, although their mechanisms of action are unclear. In addition, the release of CCK from STC-1 cells is also stimulated by the neuropeptide, bombesin, which is known to stimulate CCK secretion in vivo [19]. The stimulatory effects of DBcAMP, KC1, forskolin, /3-PMA and L-tryptophan on the release of CCK have been observed in the hormone-enriched cells isolated from canine jejunum [20,21] and duodenum (Xue, Chang and Chey, unpublished data) and rat duodenum [22]. The effects of the other agents, except for plaunotol, were observed in the CCK cell-enriched preparations isolated from the rat [22] and dog duodenum (Xue, Chang and Chey, unpublished data). Thus, STC-1 cells appear to share many characteristics with the mucosal CCK-secreting cells and should be a very good model for studying the cellular mechanism of CCK release. In addition, putting together the results of the presenti study and those of the previous ones [20-22], it may be concluded that the release of CCK from STC-1 or mucosal CCK cells involves both cAMP- and Ca2+-dependent mechanism(s). The observation that IBMX substantially enhanced the stimulatory effects of both DBcAMP and forskolin further suggests that the release of CCK from these cells is strongly modulated by cellular phosphodiesterase activity, which catalyzes the hydrolysis of cAMP. The strong stimulatory effect of 4-/3-PMA, but not its a-isomer, also suggested that cellular protein kinase C activity may be involved in the release of CCK. Although Ca 2+ and phospholipid are known to be required for the activity of protein kinase C [40,41],

346

C.H. Chang et aL / Biochimica et Biophysica Acta 1221 (1994) 339-347

the relationship among these elements of a signal transduction pathway involved in the release of CCK is not clear at present and remains to be elucidated. The results of the present study have provided some new information about the mechanism of action of certain luminal stimulants. Plaunotol, which is an antiulcer agent isolated from herbal extract, has been shown to stimulate the release of secretin and pancreatic exocrine secretion of volume, bicarbonate and enzyme in both rats [26] and humans [42]. In the present study, we have shown that plaunotol stimulates the release of CCK from STC-1 cells. Like those of forskolin and DBcAMP, the stimulatory effect of plaunotol on CCK secretion is potentiated by IBMX, suggesting that it may act via a cAMP-dependent mechanism. On the other hand, the stimulatory effect of camostat at 1 mM or higher concentrations on CCK secretion does not appear to involve a cAMP-dependent mechanism as it is not affected by IBMX. It has been proposed [10] that camostat stimulates CCK release in vivo through inhibition of proteases that act on a hypothetical CCK-releasing factor [43,44]. However, it has been documented [45] that camostat is a very strong protease inhibitor with K i ranging from 1.2. 10 -6 M for thrombin to 1.95 • 10 -9 M for trypsin. We found that camostat at 10 -4 M (a concentration capable of inhibiting proteases maximally) does not stimulate CCK secretion from either STC-1 cells (present study) or CCK cell-enriched preparation isolated from the rat [22]. We have also found that a similar protease inhibitor, phenylmethylsulfonyl fluoride (1 mM) does not stimulate the release of CCK from STC-1 cells. These observations together with the requirement of a high concentration of camostat ( > 0.5 mM) to stimulate CCK release from STC-1 cells or the rat mucosal cells suggest that camostat stimulates CCK release from these cells by an effect independent of its ability to inhibit proteases. The STC-1 cells can serve as a model for the investigation of the cellular mechanism of CCK release, because of its high number of CCK-containing cells which secrete CCK-LI in response to known CCK secretagogues. In the present study, we have shown that bombesin stimulates the release of CCK-LI from these cells. This peptide is an analog of mammalian gastrinreleasing peptide and both have been shown to stimulate secretion of CCK in vivo [18,19]. Bombesin has also been shown to stimulate both endocrine and exocrine secretions from other cell Xypes and its action is mediated via mobilization of intraceUular Ca 2÷ [46,47]. It is very likely that in STC-1 cells bombesin also stimulates the release of CCK via a Ca2÷-dependent mechanism. Therefore, these cells serve as a useful model for studying the molecular mechanism of receptor-mediated, Ca2+-dependent secretion of CCK. Moreover, bombesin has been shown to stimulate the

growth of other tumor cells [48-50] and thus may also stimulate the growth of STC-1 cells. If this is proved to be true, these cells may be a good model for studying regulation of cell growth by bombesin. The fact that these cells express and secrete CCK suggests that they also may be a useful model for studying the regulation of gene expression of CCK and the post-tranlational processing of CCK prohormones. However, it should be noted that STC-1 cells also secrete secretin and other hormones [25] so that it remains possible that some of the regulatory processes involved in secretion and gene expression of CCK in these cells may be different from the mucosal CCK cells.

5. Acknowledgements The authors wish to thank Dr. Seth Grant for providing STC-1 cells, Dr. Keiko Shiratori and Sankyo Pharmaceuticals for providing plaunotol, Brian Erway and Laura Braggins for technical assistance, and Ms. Pat Faiello for preparation of the manuscript. This study was supported by NIH grant No. 25692.

6. References [1] Jorpes, J.E. and Mutt, V. (1973) in Secretin, Cholecystokinin, Pancreozymin and Gastrin (Jorpes, J.E. and Mutt, V., eds.), pp. 1-194, Springer, Berlin. [2] Mutt, V. (1980) in Gastrointestinal Hormones (Glass, G.BJ., ed.), pp. 169-221, Raven Press, New York. [3] Rehfeld, J.F. (1989) in Handbook of Physiology, Section 6, The Gastrointestinal System, Vol. II, Neural and Endocrine Biology (Makhlouf, G.M., ed.), pp. 337-358, Oxford University Press, New York. [4] Becker, H.O., Werner, M. and Schafmayer, A. (1984) Am. J. Surg. 147, 124-129. [5] Chang, T.-M. and Chey, W.Y. (1983) Dig. Dis. Sci. 28, 456-468. [6] Shiratori, K., Chen, Y.F., Chey, W.Y., Lee, K.Y. and Chang, T.-M. (1986) Gastroenterology 91, 1171-1178. [7] Watanabe, S., Shiratori, K., Takeuchi, T., Chey, W.Y., You, C.H. and Chang, T.-M. (1986) Dig. Dis. Sci. 31, 919-924. [8] Gelin, J., Rehfeld, J.F., Jansson, R., Thronell, E. and Svanvik, J. (1986) Scand. J. Gastroenterol. 21, 235-238. [9] Maton, D.N., Selden, A.C. and Chadwick, V.S. (1984) Scand. J. Gastroenterol. 19, 831-834. [10] G6ke, B., Fenchel, K., Knobloch, S., Arnold, R., and Adler, G. (1988) Pancreas 3, 576-579. [11] Liddle, R.A., Green, G.M., Conrad, C.K. and Williams, J.A. (1986) Am. J. Physiol. 251, G243-G248. [12] Owyang, C., May, D. and Louie, D.S. (1986) Gastroenterology 91, 637-643. [13] Rhodes, R.A., Skerven, G., Chey, W.Y. and Chang, T.-M. (1988) Pancreas 3, 391-398. [14] Watanabe, S., Lee, K.Y., Chang, T.-M., Berger-Ornstein, L. and Chey, W.Y. (1988) Am. J. Physiok 254, G837-G842. [15] Li, P., Lee, K.Y., Ren, X.S., Chang, T.-M. and Chey, W.Y. (1990) Gastroenterology 98, 1642-1648. [16] Kim, C.K., Lee, K.Y., Wang, T., Sun, G., Chang, T.-M., and Chey, W.Y. (1989) Am. J. Physiol. 258, G944-G949.

C.H. Chang et al. / Biochimica et Biophysica Acta 1221 (1994) 339-347

[17] Cantor, P., Hoist, J.J., Knuthsen, S. and Rehfeld, J.F. (1986) Scand. J. Gastroenterol. 21, 1069-1072. [18] Nakano, I., Miyazaki, K., Funokoshi, A., Tateishi, K., Hamaoka, T. and Yajima, H. (1988) Regul. Pept. 23, 153-159. [19] Cuber, J.C., Vilas, F., Charles, N., Bernard, C. and Chayvialle, J.A. (1989) Am. J. Physiol. 256, G989-G996. [20] Barber, D.L., Walsh, J.H. and Soil, A.A. (1986) Gastroenterology 91, 627-636. [21] Koop, I. and Buchan, A.M.J. (1992) Gastroenterology 102, 2834. [22] Chang, C., Chang, T.-M. and Chey, W.Y. (1990) Gastroenterology 98, A486. [23] Madsen, O.D., Larssen, L.I., Rehfeld, J.F., Schwartz, T.W., Lernmark, A., Labrecque, A.D. and Steiner, D.F. (1986) J. Cell. Biol. 103, 2025-2034. [24] Aponte, G.W., Keddie, A., Hallden, G., Hess, R. and Link, P. (1991) Proc. Natl. Acad. Sci. USA 88, 5282-5286. [25] Rindi, G., Grant, S.G.N., Yiangou, Y., Ghatei, M.A., Bloom, S.R., Bautch, V.L., Soleia, E. and Polak, J.M. (1990) Am. J. Pathol. 136, 1349-1363. [26] Chang, J.-H., Watanabe, S., Shiratori, K., Moriyoshi, Y. and Takeuchi, T. (1989) Digestion 44, 142-147. [27] Sun, G., Chang, T.-M., Xue, W., Wey, J.F.Y. and Chey, W.Y. (1992) Am. J. Physiol. 262, G35-G43. [28] Winer, B.J. (1971) Statistical Principles in Experimental Design. McGraw-Hill, New York. [29] Odum, L. and Rehfeld, J.F. (1990) Biochem. J. 271, 31-36. [30] Krieger, T.J. and Hook, V.Y. (1991) J. Biol. Chem. 266, 83768373.28. [31] Tanaka, S., Nomizu, M. and Kurosumi, K. (1991) J. Histochem. Cytochem. 39, 809-821. [32] Takeuchi, T., Dickinson, C.J., Taylor, I.L. and Yamada, T. (1991) J. Biol. Chem. 266, 409-415. [33] Marino, L.R., Takeuchi, T., Dickinson, C.J. and Yamada, T. (1991) J. Biol. Chem. 266, 6133-6136.

347

[34] Johansen, E.E., O'Hare, M.M., Wultt, B.S. and Schwartz, T.W. (1991) Endocrinology 129, 553-555. [35] Yamada, T. and Chiba, T. (1989) in Handbook of Physiology, Section 6, The Gastrointestinal System (Schultz, S.G., ed.), Vol. II, Neural and Endocrine Biology (Makhiouf, G.M., ed.), Oxford University Press, New York, pp. 431-453. [36] Huttner, W.B. (1988) Annu. Rev. Physiol. 50, 363-376. [37] Hortin, G., Folz, R., Gordon, J.I. and Straus, A.W. (1986) Biochem. Biophys. Res. Commun. 141, 326-333. [38] Bateman, A., Solomon, S. and Bennett, H.P.J. (1990) J. Biol. Chem. 265, 22130-22136. [39] Larsson, L.I. and Rehfeid, J.F. (1981) Science 213, 768-770. [40] Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., Nishizuka, Y., Tamura, A. and Fujii, T.J. (1979) Biochemistry 86, 575-578. [41] Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. and Nishizuka, Y. (1979) J. Biol. Chem. 3692-3695. [42] Shiratori, K., Watanabe, S.I., Takeuchi, T., Chang, J.H. and Moriyoshi, Y. (1989) Pancreas 4, 323-328. [43] Lu, L., Louie, D. and Owyang, C.H. (1989) Am. J. Physiol. 256, G430-G435. [44] Miyasaka, K., Guan, D., Liddle, R.A. and Green, G.M. (1989) Am. J. Physiol. 257, G175-G181. [45] Tamura, Y., Hirado, M., Okamura, K., Minato, Y. and Fujii, S. (1977) Biochim. Biophys. Acta 484, 417-422. [46] Jensen, R.T., Moody, T., Pert, C., Rivier, J.G. and Gardner, J.D. (1978) Proc. Natl. Acad. Sci. USA 75, 6139-6143. [47] Brown, K.D., Biay, J., Irvine, R.F., Heslop, J.P. and Berridge, M.J. (1984) Biochem. Biophys. Res. Commun. 123, 377-384. [48] Rozengurt, E. and Sinnett-Smith, J. (1983) Proc. Natl. Acad. Sci. USA 80, 2936-2940. [49] Willey, J.C., Lechner, J.F. and Harris, C.C. (1984) Exp. Cell Res. 153, 245-248. [50] Cuttitta, F., Carney, D.N., Mulshine, J., Moody, T.W., Fedorko, J., Fischler, A. and Minna, J.D. (1985) Nature 316, 823-826.