JOURNAL OF CELLULAR PHYSIOLOGY 15O:l-7 (1992)
Receptor-MediatedAutocrine Growth-Stimulatory Effect of 5-Hydroxytryptamine on Cultured Human Pancreatic Carcinoid Cells JIN ISHIZUKA, R. DANIEL BEAUCHAMP, COURTNEY M. TOWNSEND, JR.,* GEORGE H. GREELEY, JR., AND JAMES C. THOMPSON Department of Surgery, The University of Texas Medical Branch, Calveston, Texas 77550 5-hydroxytryptamine (5-HT) i s a mitogen for fibroblasts, vascular smooth muscle cells, renal mesangial cells, and jejunal crypt cells. The human carcinoid cell line (termed BON) that we established in our laboratory from a pancreatic carcinoid tumor produces and secretes 5-HT. In this study, therefore, we examined the effect of 5-HT on growth of BON cells. Furthermore, by use of selective 5-HT receptor antagonists, we examined receptor and post-receptor mechanisms by which 5-HT-induced responses were produced. 5-HT stimulated growth of BON cells. 5-HT stimulated phosphatidylinositol (PI) hydrolysis in a dose-dependent fashion and inhibited cyclic AMP production in a dose-dependent fashion. The 5-HT,,, receptor antagonist, SDZ 21 -009, prevented the reduction of cyclic AMP production evoked by 5-HT and inhibited the mitogenic action of 5-HT. The 5-HT,., receptor antagonist, mesulergine, competitively inhibited PI hydrolysis, but did not affect the mitogenic action of 5-HT. The mitogenic action of 5-HT and the reduction of cyclic AMP production evoked by 5-HT were also inhibited by pertussis toxin. These results suggest that 5-HT is an autocrine growth factor for BON cells and that mitogenic mechanism of 5-HT involves receptor-mediated inhibition of the production of cyclic AMP which may be linked to pertussis toxin-sensitive GTP binding protein. 8-bromo-cyclic AMP inhibited growth of BON cells whereas 8-bromo-cyclic CMP had no effect on cell growth. Involvement of protein kinase A in BON cell growth regulation was confirmed by the observation that a CAMP-dependent protein kinase antagonist (Rp-CAMPS)could stimulate BON cell growth.
5-hydroxytryptamine (5-HT) or serotonin, known as culture for more than four Years (Lawrence et al., a neurotransmitter and vasoactive agent, is produced 1990). These carcinoid cells produce and secrete 5-HT and contained in several tissues throughout the body, (Lawrence et al., 1990). We find that 5-HT has an including the gastrointestinal enterochromaffin cells autocrine growth effect for BON cells. In order to (Sjolund et al., 1983), central nervous system (Shields differentiate which specific receptor and post-receptor and Eccleston, 19731, and platelets (Marmaras and second messenger systems mediate the autocrine effect Mimikos, 1971). Among the numerous actions of 5-HT, of 5-HT on growth of BON cells, selective receptor recent studies suggest that 5-HT has a growth-stimu- antagonists, SDZ 21-009 (5-HT,, and 5-HT,, receptor latory activity for Chinese hamster lung fibroblasts antagonist) and mesulergine (CU 32-085 [5-HTIc and (Seuwen et al., 19881, bovine vascular smooth muscle 5-HT, receptor antagonist]) have been used. Phosphaticells (Nemecek et al., 1986), rat renal mesangial cells dylinositol (PI) and CAMPproduction have been mea(Takuwa et al., 19891, and rat jejunal crypt cells (Tut- sured in response to 5-HT or the antagonists. ton and Barkla, 1986). MATERIALS AND METHODS Overproduction of 5-HT is often observed in patients The following substances were purchased: Dulbeco’s with carcinoid tumors (Lembeck, 1953; Thorson et al., 1954; Mazzaferri and O’Dorisio, 1987). The ef- modified Eagle’s medium (DMEM) and F-12K medium fects of 5-HT on several organs, including gastrointes- (Gibco Laboratories, Grand Island, NY); fetal calf setinal tract, have been studied (Jaffe, 1979) in these rum (FCS) (Hyclone Laboratories, Inc., Logan, UT); and cyclic AMP radiopatients. Although 5-HT is mitogenic for selected cell 3H-thymidine, my~-~H-inositol, types in other species, potential autocrine growth- immunoassay (RIA) kit (Amersham Corporation, Arregulatory effects on human carcinoid tumor cells have lington Heights, IL); Dowex AG-1 x 8 chromatography not been studied because of the lack of a n ideal experimental model. We established the functioning human carcinoid cell line (termed BON) in tissue culture from Received March 26, 1991; accepted August 13, 1991. a pancreatic carcinoid tumor and have maintained it in *To whom reprint requestsicorrespondence should be addressed. C 1992 WILEY-LISS. INC
2
ISHIZUKA ET AL
(formate form) (Bio-Rad Laboratories, Richmond, CAI; other chemicals including 5-HT, ascorbic acid, bovine serum albumin (BSA, RIA grade), isobutylmethylxanthine (IBMX), LiC1, pertussis toxin, 8-bromo-cyclic AMP, 8-bromo-cyclic GMP (Sigma Chemicals, St. Louis, MO), and Rp-adenosine-3’,5’-cyclic monophosphothioate (Rp-CAMPS)(BIOLOG, La Jolla, CAI. Selective 5-HT receptor antagonists, SDZ 21-009 and mesulergine, were kindly supplied by Sandoz Pharma Ltd. (Basel, Switzerland). Concentrations of these 5-HT receptor antagonists employed in this study were selected based on antagonistic efficacies for 5-HT receptors which were provided by Sandoz Pharma. Efficacy and selectivity of SDZ 21-009 has been determined by bioassay methods of Schoeffter et al. (1988) and Bouhelal et al. (1988), and efficacy and selectivity of mesulergine has been determined by bioassay methods of Hoyer et al. (1985) and Doyle et al. (1986). Except for 8-bromo-cyclic AMP and IBMX, all reagents were dissolved with double distilled water. 8-bromo-cyclic AMP was dissolved with 0.01 N NaOH and IBMX was dissolved with 0.2 N NaOH. The above solvents were used appropriately as control vehicles in the present experiments. All experiments were performed using six dishes or wells each time and mean c SEM of a t least three different experiments were shown.
BON cell culture BON cells have been maintained in DMEM and F-12K (1:l) media, containing 5% FCS in a humidified atmosphere of 95% air and 5% COz at 37°C. Stock cultures have media changed every three days (doubling time is approximately 48 hours). In order to compare results between experiments, BON cells in passages 9-13 were utilized in the present study. Effect of 5-HT and 5-HT receptor antagonists on 3H-thymidineincorporation BON cells (3 x lo5) grown in DMEM and F-12K (1:l) containing 5% FCS were rendered quiescent by complete deprivation of serum for 24 hours. Then a DMEMi F-12K containing 3H-thymidine (1Ciiml) and reagents, 5-HT (lo-’ to M) alone or with SDZ 21-009 M) or mesulergine (lop7M), were added. After another 24 hours, cell monolayers were fixed with 5% trichloroacetic acid (TCA) and washed twice. Acid-insoluble material was collected with 0.1 N NaOH and counted by a liquid scintillation spectrometry. Ascorbic acid (0.01%), which prevents degradation of 5-HT, was added to solutions of all reagents, including vehicles for control, in this experiment and following all experiments. Effect of 5-HT and 5-HT receptor antagonists on cell growth BON cells (1 x lo5)grown in DMEM/F-lBK containing 5% FCS had medium replaced by the same culture medium containing 0.5% FCS to examine the stimulatory effect of 5-HT on cell growth. After two days in culture, 5-HT (lop7, and lop5 M), SDZ 21-009 (lo-’ to M), and mesulergine M) were added alone or in combination every other day. Medium was replaced every other day. Every other day, cells to be counted were detached from dishes with trypsin (1:250,
Gibco) and diluted tenfold with Isoton (Curtin Matheson Scientific, Houston, TX) to stop the action of trypsin. Then, cell number was counted by a Coulter counter which was electrically set up to count cells under 100 p,m in size. Cell number as determined by a Coulter counter was similar (less than 5% difference) to viable cell number determined by a dye (trypan blue) exclusion method using a hemocytometer. Also, coefficient variances of low and high counts determined by this electric counter were less than 2%.
Effect of 5-HT and 5-HT receptor antagonists on PI hydrolysis PI hydrolysis (inositol monophosphate formation [IP]) was measured following a protocol adapted from Hawkins et al. (1989). In brief, BON cells (3 x lo5), which were harvested by trypsin (1:250) and cultured in 24-well tissue culture plate for 2 days, were incubated with DMEMIF-12K containing 5% FCS supplemented with my~-~H-inositol (10 p-Ci/ml) for 16 hours. Cells were then incubated for 10 min with oxygenated Krebs-Ringer bicarbonate buffer (KRBB, pH 7.4) containing HEPES (10 mM), BSA (0.1%), glucose (2.5 mM), and LiCl(10 mM). After another 15-min incubation in the presence of 5-HT (lo-’ to lop4 MI alone or with mesulergine (lop7 M) or SDZ 21-009 M), cells were extracted with 10% perchloric acid and immediately neutralized with 6 N KOH. A fraction of I P was extracted by anion exchange chromatography (Dowex AG-1 x 8; formate form; 2 0 0 4 0 0 mesh) using a mixture of formic acid (0.1 M) and ammonium formate (0.2 M) as an eluent. Radioactivity of IP fraction was measured by a liquid scintillation counter. Effect of 5-HT, 5-HT receptor antagonists, or pertussis toxin on the production of cyclic AMP BON cells (5 x lo5),which were harvested by trypsin and cultured in 35 x 10 mm tissue culture dishes for 2 days, were incubated in oxygenated KRBB (pH 7.4) containing HEPES (10 mM), BSA (0.1%), glucose (2.5 mM), and IBMX (1mM). After a 30-min incubation in the presence of 5-HT (lo-’ to M) alone or with SDZ 21-009 (lop7 M) or mesulergine M), cells were extracted with 5% TCA and neutralized with a n excess of CaCO, just before RIA (Tihon et al., 1977). After acetylation of samples with acetic anhydride and triethylamine, intracellular cyclic AMP levels were measured by cyclic AMP RIA kit. To examine the effect of pertussis toxin on cyclic AMP production in BON cells, BON cells were preincubated for 5 hours with or without pertussis toxin (100 ngiml), and then cyclic AMP production in response to 5-HT (lo-’ to M) was examined. Effect of 8-bromo-cyclicAMP or 8-bromo-cyclic GMP on cell growth BON cells (5 x lo4) grown in DMEMIF-12K containing 5%FCS were placed in culture medium containing 0.5% FCS. Forty-eight hours later, 8-bromo-cyclic AMP (lop4M) alone or with 5-HT M), or 8-bromo-cyclic GMP M) alone was added every other day. Culture medium was changed every other day and cell number was counted as described in a previous section.
3
AUTOCRINE EFFECT OF 5-HYDROXYTRYPTAMINE
Effect of Rp-CAMPSon cell growth BON cells (5 x lo4)grown in DMEM/F-lBK containing 5% FCS were placed in culture medium containing 0.5% FCS. Forty-eight hours later, selective cyclic AMP-dependent protein kinase antagonist, Rp-CAMPS (5 x MI, was added every other day. Culture medium was changed every other day and cell number was counted as described in a previous section. Effect of pertussis toxin on 5-HT-stimulated cell growth BON cells (5 x lo4)grown in DMEM/F-l2K containing 5% FCS were placed in culture medium containing 0.5% FCS. Forty-eight hours later, 5-HT (lop5 M) alone, pertussis toxin (100 ng/ml) alone, or 5-HT M) with pertussis toxin (10, 50, and 100 ng/ml) were added every other day. Culture medium was changed every other day and cell number counted. Statistical analysis Data from each study were analyzed by unpaired t-test and were shown as mean 5 SEM of at least three experiments. Significance was assumed for P value less than 0.05.
CI
n
* *
13
I
Effect of 5-HT and 5-HT receptor antagonists on cell growth 5-HT stimulated growth of BON cells in a dosedependent fashion with maximum effect at M (data not shown). The growth-stimulatory effect of 5-HT was completely inhibited by SDZ 21-009 (lop7M), whereas mesulergine had no effect (data not shown). The inhibitory effect of SDZ 21-009 on 5-HT-stimulated growth of BON cells was dose-dependent (data not shown). SDZ 21-009 alone also significantly inhibited the growth of BON cells, while mesulergine alone did not affect the growth of BON cells (Fig. 2). The inhibitory effect of SDZ 21-009 did not persist after cessation of treatment and cell growth recovered rapidly (data not shown). Effect of 5-HT and 5-HT receptor antagonists on PI hydrolysis 5-HT stimulated PI hydrolysis in BON cells in a biphasic dose-dependent fashion and a peak response of PI hydrolysis was observed a t M of 5-HT (Fig. 3). Mesulergine (lop7M) inhibited PI hydrolysis stimulated by 5-HT in a competitive fashion. Thus, the peak response shifted from lop7 M to M of 5-HT. On the other hand, SDZ 21-009 (lop7M) did not affect PI hydrolysis stimulated by 5-HT.
* *
0
c
X
* *
;1 1 0
v
cc I TI -
1 n
9
7
CONTROL
7
8
5
6
4
5-HYDROXYTRYPTAMINE (-LOG M)
Fig. 1. Effects of 5-HT and 5-HT receptor antagonists on 3Hthymidine incorporation of BON cells. In this figure and following figures, error bars indicate standard error of the mean of at least three separate experiments. 0: 5-HT added alone. B: 5-HT with SDZ 21-009 M). * indicatesP < 0.05 vs. M). B: 5-HT with mesulergine control; + indicates P < 0.05 vs. 5-HT alone.
12 -
14
RESULTS Effect of 5-HT and 5-HT receptor antagonists on 3H-thymidineincorporation 5-HT stimulated 3H-thymidine incorporation in serum-deprived BON cells in a dose-dependent fashion / ~ B antagonist, SDZ (Fig. 1).Selective ~ - H T ~ Areceptor 21-009, completely inhibited 3H-thymidine incorporation stimulated by 5-HT, while selective 5-HT,c,z receptor antagonist, mesulergine, failed to affect 3Hthymidine incorporation stimulated by 5-HT.
* *
I2
'z
10
*
-
*
X Y
rK
w
m
3 6
Z
i
w
4
0
2
0
0
2
4
6
DAYS
Fig. 2. Effect of 5-HT and 5-HT receptor antagonists on growth of BON cells. 0: DMEMIF-12 K (1:l)alone. .: 5-HT MI alone. a: SDZ 21-009 M) alone. B:5-HTwith SDZ 21-009. 0: mesulergine M) alone. The day when treatments were started was designated as day 0. * indicates P < 0.05 vs. DMEMIF-12 K alone; + indicates P < 0.05 vs. 5-HT alone.
Effect of 5-HT, 5-HT receptor antagonists, or pertussis toxin on the production of cyclic AMP 5-HT inhibited the production of cyclic AMP in BON cells in a dose-dependent fashion, and SDZ 21-009 Completely prevented the reduction of cyclic AMP production evoked by 5-HT (Fig. 4a). On the other hand, mesulergine did not affect the reduction of cyclic AMP production evoked by 5-HT. SDZ 21-009 alone slightly increased basal CAMPlevel, while mesulergine alone slightly decreased it; however, both effects were not statistically significant (data not shown). Pertussis toxin completely prevented the reduction of cyclic AMP production evoked by 5-HT (Fig. 4b). Per-
ISHIZUKA ET AL
* *1 * *
120
11 :*:
100 -1
2 Iz
00
80
LL 0
Be
60
*
Y
5
4
*
4c CONTROL
a
7 6 5 5-HYDROXYTRYPTAMINE (-LOG M)
4
Fig. 3. Effect of 5-HT alone or with mesulergine or SDZ 21-009 on PI hydrolysis in BON cells. Accumulation of inositol monophosphate (IP) in BON cells was used as a marker of PI hydrolysis. 0:5-HT alone. .: 5-HT with mesulergine M). R : 5-HT with SDZ 21-009 M). Mesulergine or SDZ 21-009 was added 10 min before 5-HT. * indicates P < 0.05 vs. control.
CONTROL
9
8
a
Effect of Rp-CAMPSon cell growth The selective cyclic AMP-dependent protein kinase antagonist, Rp-CAMPS (5 x lop5 MI, significantly stimulated the growth of BON cells (Fig. 7). Effect of pertussis toxin on 5-HT-stimulated cell growth Pertussis toxin alone (100 ng/ml) inhibited the growth of BON cells, and inhibited the growth of BON cells stimulated by 5-HT in a dose-dependent fashion (Fig. 8). The inhibitory effect of pertussis toxin did not persist after cessation of treatment and cell growth recovered rapidly (data not shown). DISCUSSION We have shown in this study that 5-HT is a n autocrine growth factor for human carcinoid tumor cells. Furthermore, the present results indicate that: 1)functioning human pancreatic carcinoid cells have at least two different 5-HT receptors (5-HTIA,,, and 5-HTlc,4; 2) inhibition of cyclic AMP production by 5-HT is mediated through 5-HTlA or 5-HT1, receptors; 3) activation of PI hydrolysis by 5-HT is mediated through 5-HTlc or 5-HT2 receptors; 4) the autocrine growth effect is transmitted by 5-HTIA or 5-HTIB receptors,
6
5-HYDROXYTRYPTAMINE (-LOG M)
1
110, 100
* \ o * 0 ,‘* *
tussis toxin alone slightly increased basal CAMPlevel; however, its effect was not statistically significant (data not shown).
Effect of 8-bromo-cyclicAMP or 8-bromo-cyclic GMP on cell growth 8-bromo-cyclic AMP M) significantly inhibited the growth of BON cells (Fig. 5a), while 8-bromo-cyclic GMP M) did not affect the growth of BON cells (Fig. 5b). Furthermore, 8-bromo-cyclic AMP ( M) prevented the growth-stimulatory effect of 5-HT M) (Fig. 6). The inhibitory effect of 8-bromo-’cyclicAMP did not persist after cessation of treatment and cell growth recovered rapidly (data not shown).
7
* 1
9 ,
CONTROL
b
9
8
7
6
5
4
5-HYDROXYTRYPTAMINE (-LOG M)
Fig. 4. a: Effect of 5-HT alone (0) or with SDZ 21-009 (0) or mesulergine ( A ) on the production of cyclic AMP in BON cells. b Effect of 5-HT alone ( 0 )or with pertussis toxin (PT) (100 wgiml) ( 0 ) on the production of cyclic AMP in BON cells. Results were expressed as a percent of control. * indicates P < 0.05 vs. control.
activation of which decreases cyclic AMP production apparantly through a pertussis toxin-sensitive inhibitory GTP binding protein; and 5) increase of intracellular cyclic AMP level leads to the inhibition of cell growth, while inhibition of cyclic AMP-dependent protein kinase leads to the stimulation of cell growth. As reported previously (Lawrence et al., 19901, BON cells secrete 5-HT. With a short-term incubation study, we have found that BON cells secrete 5-HT, 0.92 5 0.10 ng/ml/30 min (2.38 x lo-’ M) and 2.24 ? 0.12 ng/ml/60 min (5.78 x lo-’ M) in a serum-free condition (Ishizuka et al., 1990), in which BON cells grow very slowly (doubling time, 144 hours). These data may suggest that BON cells secrete and accumulate, probably in a time- and serum-dependent manner, a sufficient amount of 5-HT which stimulates growth of BON cells in a n autocrine fashion. Previously reported growth-stimulatory effects of 5-HT on non-transformed Chinese hamster lung fibroblasts appear to be produced by inhibition of cyclic AMP production, and this effect is mediated by 5-HTlB receptors (Seuwen et al., 1988). Induction of DNA synthesis by 5-HT in bovine aortic smooth muscle cells
AUTOCRINE EFFECT OF 5-HYDROXYTRYPTAMINE
12-
14-
A
-10-
'
v)
I
!n
I
x
5
1210-
8-
X
W
v
5m
I 3
z
6I
J
- 1 4 W
0
2 0
2
0
B
DAYS Fig. 6. Effect of 8-bromo-cyclic AMP on 5-HT-stimulated growth of BON cells. 0 : DMEMIF-12 K (1:l)alone. . :5-HT M) alone. N: 5-HT with 8-bromo-cyclic AMP (10- MI. The day when treatments were started was designated a s day 0. * indicates P < 0.05 vs. DMEMIF-12 K alone; + indicates P < 0.05 vs. 5-HT alone.
* 2
6
4
*
8
DAYS Fig. 5. A Effect of 8-bromo-cyclic AMP on growth of BON cells. 0: DMEMIF-12 K (1:l) alone. t3:8-bromo-cyclic AMP Mj. B: Effect of 8-bromo-cyclic GMP on growth of BON cells. 0: DMEMIF-12 K (1:l) alone. El: 8-bromo-cyclic GMP MI. The day when treatments were started was considered as day 0. * indicates P < 0.05 vs. DMEMIF-12 K alone.
Y a
m
x 3 z W
0
4
.1
*
0
4
6
8
DAYS
also occurs through a pertussis toxin-sensitive pathway, but is not linked to alterations in cyclic AMP levels (Kavanaugh et al., 1988). The findings of our study suggest that the growth-stimulatory effect of 5-HT on BON cells is mediated through mechanisms similar to those described for Chinese hamster lung fibroblasts (Seuwen e t al., 1988). In the present study, we used relatively selective 5-HT receptor antagonists. SDZ 21-009, thus, antagonizes both 5-HTlA and 5-HTlB receptors. Previous reports have suggested that activation of either 5-HTl, or 5-HTlB receptors inhibits the production of cyclic AMP (Schoeffter and Hoyer, 1988; Bouhelal et al., 1988). On the other hand, there is a consensus that in bovine or human cells, 5-HTlB receptor is never present; however, a pharmacologically different but functionally equivalent receptor, 5-HTlD, may be expressed instead of 5-HTlB receptor (Hoyer and Middlemiss, 1989). Furthermore, activation of the 5-HTlD receptor has been shown to inhibit adenylate cyclase (Schoeffter et al., 1988; Hoyer and Scheoffter, 1988). The effects of the membvane-permeable synthetic cyclic AMP analogue, 8-bromo-cyclic AMP, that significantly inhibited growth of BON cells support the conclusion that 5-HT stimulates growth of BON cells by inhibiting the
Fig. 7. Effect of Rp-CAMPSon growth of BON cells. 0 : DMEMIF-12 M). The day when treatment K (1:lj alone. 8 :Rp-CAMPS (5 x with Rp-CAMPS was started was considered a s day 0. * indicates P < 0.05 vs. DMEMIF-12 K alone.
production of cyclic AMP. Taken together, since SDZ 21-009 cannot antagonize 5-HTlD receptor in the concentrations employed in the present study (based on the information provided by Sandoz Pharmaceutical), the inhibitory effect on the production of cyclic AMP in BON cells is most likely mediated through 5-HT1, receptor. The selective cyclic AMP-dependent protein kinase antagonist, Rp-CAMPS,stimulated the growth of BON cells. This antagonist inhibits the activated cyclic AMP-dependent protein kinase (Pereira et al., 1987; Erneux et al., 1986); therefore, the finding that RpCAMPS alone stimulates BON cell growth suggests that cyclic AMP-dependent protein kinase in BON cells is constantly stimulated in tissue culture and maintains a tonic negative control of BON cell growth. High concentrations of cyclic AMP, as used in the
ISHIZUKA ET AL
6 *
5 m '
z
#
ACKNOWLEDGMENTS The authors would like to thank Jell Hsieh and Mary Schmitz-Brown for their technical assistance and Kelly Lee, Sharon Llewellyn, and Cathy Gehret for their help in the preparation of this manuscript. Supported by grants from the National Institutes of Health (5R37 DK 15241, PO1 DK 35608,5Kll CA 01309) and by a grant from the American Cancer Society (PDT-220).
4
x v r Y 3 W
m
I 2
2
2
3 W 0
tients with carcinoid tumor may also inhibit growth of carcinoid tumor cells.
1
LITERATURE CITED 0 0
2
4
DAYS
Fig. 8. Effect of pertussis toxin (PT) on 5-HT-stimulated growth of BON cells. 0 : DMEMIF-12 K (1:l) alone. m: 5-HT M) alone. B:PT (100 ngiml) alone. El:5-HT with PT (100 ngiml). 69: 5-HT with PT (50 ngiml). B: 5-HT with PT (10 ngiml). The day when treatments were started was designated as day 0. * indicates P < 0.05 vs. DMEMIF-12 K alone; # indicates P < 0.05 vs. 5-HT alone.
present study, can activate cyclic GMP-dependent protein kinase (Lincoln and Corbin, 1983), which may have growth-regulatory effects (Corbin et al., 1990; Garg and Hassid, 1989). In the present study, 8-bromocyclic GMP did not show any inhibitory effect on the growth of BON cells. These results support the notion that the cyclic AMP pathway is, at least in part, responsible for growth-regulation in BON cells, but cyclic GMP does not have a similar effect. Pertussis toxin inhibited 5-HT-stimulated growth of BON cells and prevented the inhibition of CAMPproduction evoked by exogenous 5-HT. Since pertussis toxin induces ADP-ribosylation of the inhibitory GTP binding protein leading to its functional uncoupling from adenylate cyclase (Ui, 1984), our results suggest that the 5-HT receptors involved in growth-regulatory mechanism in BON cells are coupled to a pertussis toxin-sensitive inhibitory GTP binding protein. In addition to the observations that both SDZ 21-009 and pertussis toxin inhibited growth of BON cells stimulated by 5-HT, we found that addition of either SDZ 21-009 or pertussis toxin in the absence of exogenous 5-HT resulted in inhibition of BON cell growth. These findings suggest that both agents inhibit the growthstimulatory effect of 5-HT that is endogenously released by BON cells; endogenous 5-HT released from BON cells is a n autocrine growth factor for BON cells. Somatostatin or its analogue has been known to inhibit synthesis or release of 5-HT in patients with carcinoid tumor and also to retard tumor growth in some carcinoid patients (Kvols, 1989). These facts may support our notion that 5-HT is an autocrine growth factor for carcinoid tumor cells. 5-HT may play a n important role in pathophysiological aspects of carcinoid tumor in vivo by endocrine, paracrine, and potentially autocrine actions. Although there have been several reports that 5-HT receptor antagonists can improve clinical symptoms related to overproduction of 5-HT (Anderson et al., 1987; Ahlman et al., 19851, our findings suggest that administration of proper selective 5-HT receptor antagonists to pa-
Ahlman, H., Dahlstrom, A,, Gronstad, K., Tisell, L.E., Oberg, K., Zinner, M.J., and Jaffe, B.M. (1985) The pentagastrin test in the diagnosis of the carcinoid syndrome. Ann. Surg., 201:81-86. Anderson, J.V., Coupe, M.O., Morris, J.A., Hodgson, H.J.F., and Bloom, S.R. (1987) Remission of symptoms in carcinoid syndrome with a new 5-hydroxytryptamine M receptor antagonist. Br. Med. J., 294:1129 (abst.). Bouhelal, R.,Smounya, L., and Bockaert, J . (1988) 5-HTI, receptors are negatively coupled with adenylate cyclase in rat substantia nigra. Eur. J. Pharmacol., 151r189-196. Corbin, J.D., Thomas, M.K., Wolfe, L., Shabb, J.B., Woodford, T.A., and Francis, S.H. (1990) New insights into cGMP action. In: The Biology and Medicine of Signal Transduction. Y. Nishizuka, M. Endo, and C. Tanaka, eds. Raven Press, New York, pp. 411-418. Doyle, V.M., Creba, J.A., Ruegg, U.T., and Hoyer, D. (1986) Serotonin increases the production of inositol phosphates and mobilizes calcium via the 5-HT2 receptor in A7r5 smooth muscle cells. Naunyn Schmiedebergs Arch. Pharmacol., 333:98-103. Erneux, C., Van Sande, J., Jastorff, B., and Dumont, E. (1986) Modulation of cyclic AMP action in the dog thyroid by its agonist and antagonist Sp- and Rp-adenosine 3',5'-monophosphorothioate. Biochem. J.,234:193-197. Garg, U.C., and Hassid, A. (1989) Inhibition of rat mesangial cell mitogenesis by nitric oxidegenerating vasodilators. Am. J . Physiol., 257:F60-F66. Hawkins, D., Enyedi, P., and Brown, E. (1989) The effects of high extracellular Ca2+and Mg2+concentrations on the levels of inositol 1,3,4,5-tetrakisphosphatein bovine parathyroid cells. Endocrinology, 124:838-844. Hoyer, D., Emgel, G., and Kalkman, H.O. (1985) Molecular pharmacology of 5-HT, and 5-HT, recognition sites in rat and pig brain membranes: Radioligand binding studies with L3Hl 5-HT, ["HI 8-OH-DPAT, (-) [lz5I] iodocyanopindolol, r3HI mesulergine and f3HJ ketanserin. Eur. J . Pharmacol., 118:13-23. Hoyer, D., and Middlemiss, D.N. (1989) Species differences in the pharmacology of terminal 5-HT autoreceptors in mammalian brain. TiPS 10:130-132. Hoyer, D., and Scheoffter, P. 11988) 5-HT,, receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in calf substantia nigra. Eur. J. Pharmacol., 147:145-147. Ishizuka, J., Parekh, D., Karp, G.W., Haber, B., Greeley, G.H., Jr., Townsend, C.M., Jr., and Thompson, J.C. (1990) Evidence of a cholinergic receptor on long-term cultured human pancreatic carcinoid cells. Gastroenterology, 98266 (abst.). Jaffe, B.M. (1979) Prostaglandins and serotonin: Nonpeptide diarrheogenic hormones. World J. Surg. 3565-578. Kavanaugh, W.M., Williams, L.T., Ives, H.E., and Coughlin, S.R. (1988) Serotonin-induced deoxyribonucleic acid synthesis in vascular smooth muscle cells involves a novel, pertussis toxin-sensitive pathway. Mol. Endocrinol., 123599-605, Kvols, L.K. (1989) Therapeutic considerations for the malignant carcinoid syndrome. Acta Oncologica, 28:433438. Lawrence, J.P., Ishizuka, J., Harber, B., Townsend, C.M., Jr., and Thompson, J.C. (1990) The effect of somatostatin on 5-hydroxytryptamine release from a carcinoid tumor. Surgery, 108;11311135. Lembeck, F. (1953) 5-Hydroxytryptamine in a carcinoid tumour. Nature, 172t910-911. Lincoln, T.M., and Corbin, J.D. (1983)Characterization and biological role of the cGMP-dependent protein kinase. In: Advances in C&dic Nucleotide Research. P. Greengard and G.A. Robison, eds. Raven Press, New York, Vol. 15, pp. 139-192. Marmaras, V.J., and Mimikos, N. (1971) Enzymic formation of serotonin in mammalian blood platelets and red cells. Experientia, 27,196-197.
AUTOCRINE EFFECT OF 5-HYDROXYTRYPTAMINE
Mazzaferri, E.L., and O’Dorisio, T.M. (1987) Introduction: Endocrine tumors-special problems in diagnosis and management. Semin. Oncol., 14:235-236. Nemecek, G.M., Coughlin, S.R., Handley, D.A., and Moskowitz, M.A. (1986) Stimulation of aortic smooth muscle cell mitogenesis by serotonin. Proc. Natl. Acad. Sci. U.S.A., 83t674-678. Pereira, M.E., Segaloff, D.L., Ascoli, M., and Eckstein, F. (1987) Inhibition of choriogonadotropin-activated steroidogenesis in cultured leydig tumor cells by the Rp diastereoisomer of adenosine 3’3’-cyclic phosphorothioate. J. Biol. Chem., 262t6093-6100. Schoeffter, P., and Hoyer, D. (1988) Centrally acting hypotensive agents with affinity for 5-HT,, binding sites inhibit forskolinstimulated adenylate cyclase activity in calf hippocampus. Br. J. Pharmacol., 95t975-985. Schoeffter, P., Waeber, C., Palacios, J.M., and Hoyer, D. (1988) The 5-hydroxytryptamine5-HTIDreceptor subtype is negatively coupled to adenylate cyclase in calf substantia nigra. Arch. Pharmacol., 337t602-608. Seuwen, K., Magnaldo, I., and Pouyssegur, J. (1988) Serotonin stimulates DNA synthesis in fibroblasts acting through 5-HT,, receptors coupled to a GI-protein.Nature, 335t254-256. Shields, P.J., and Eccleston, D. (1973) Evidence for the synthesis and
7
storage of 5-hydroxytryptamine in two separate pools in the brain. J. Neurochem., 20:881-888. Sjolund, K., Sanden, G., Hakanson, R., and Sundler, F. (1983) Endocrine cells in human intestine: An immunocytochemical study. Gastroenterology, 85:1120-1130. Takuwa, N., Ganz, M., Takuwa, Y ., Sterzel, R.B., and Rasmussen, H. (1989) Studies of the mitogenic effect of serotonin in rat renal mesangial cells. Am. J. Physiol., 257tF431-F439. Thorson, A., Biorck, G., Bjorkman, G., and Waldenstrom, J. (1954) Malignant carcinoid of the small intestine and metastases to the liver, valvular disease of the right heart (pulmonary stenosis and tricuspid regurgitation without septa1 defect),peripheral vasomotor symptoms, bronchoconstriction and unusual type of cyanosis. Am. Heart J., 47:795-817. Tihon, C., Goren, M.B., Spitz, E., and Rickenberg, H.V. (1977) Convenient elimination of trichloroacetic acid prior to radioimmunoassay of cyclic nucleotides. Analyt. Biochem., 80r652-653. Tutton, P.J.M., and Barkla, D.H. (1986)Serotonin receptors influencing cell proliferation in the jejunal crypt epithelium and in colonic adenocarcinomas. Anticancer Res., 6t1123-1126. Ui, M. (1984) Islet activating protein, pertussis toxin: A probe for functions of the inhibitory guanine nucleotide regulatory component of adenylate cyclase. Trends Pharmacol. Sci., 5277-279.
Receptor-MediatedAutocrine Growth-Stimulatory Effect of 5-Hydroxytryptamine on Cultured Human Pancreatic Carcinoid Cells JIN ISHIZUKA, R. DANIEL BEAUCHAMP, COURTNEY M. TOWNSEND, JR.,* GEORGE H. GREELEY, JR., AND JAMES C. THOMPSON Department of Surgery, The University of Texas Medical Branch, Calveston, Texas 77550 5-hydroxytryptamine (5-HT) i s a mitogen for fibroblasts, vascular smooth muscle cells, renal mesangial cells, and jejunal crypt cells. The human carcinoid cell line (termed BON) that we established in our laboratory from a pancreatic carcinoid tumor produces and secretes 5-HT. In this study, therefore, we examined the effect of 5-HT on growth of BON cells. Furthermore, by use of selective 5-HT receptor antagonists, we examined receptor and post-receptor mechanisms by which 5-HT-induced responses were produced. 5-HT stimulated growth of BON cells. 5-HT stimulated phosphatidylinositol (PI) hydrolysis in a dose-dependent fashion and inhibited cyclic AMP production in a dose-dependent fashion. The 5-HT,,, receptor antagonist, SDZ 21 -009, prevented the reduction of cyclic AMP production evoked by 5-HT and inhibited the mitogenic action of 5-HT. The 5-HT,., receptor antagonist, mesulergine, competitively inhibited PI hydrolysis, but did not affect the mitogenic action of 5-HT. The mitogenic action of 5-HT and the reduction of cyclic AMP production evoked by 5-HT were also inhibited by pertussis toxin. These results suggest that 5-HT is an autocrine growth factor for BON cells and that mitogenic mechanism of 5-HT involves receptor-mediated inhibition of the production of cyclic AMP which may be linked to pertussis toxin-sensitive GTP binding protein. 8-bromo-cyclic AMP inhibited growth of BON cells whereas 8-bromo-cyclic CMP had no effect on cell growth. Involvement of protein kinase A in BON cell growth regulation was confirmed by the observation that a CAMP-dependent protein kinase antagonist (Rp-CAMPS)could stimulate BON cell growth.
5-hydroxytryptamine (5-HT) or serotonin, known as culture for more than four Years (Lawrence et al., a neurotransmitter and vasoactive agent, is produced 1990). These carcinoid cells produce and secrete 5-HT and contained in several tissues throughout the body, (Lawrence et al., 1990). We find that 5-HT has an including the gastrointestinal enterochromaffin cells autocrine growth effect for BON cells. In order to (Sjolund et al., 1983), central nervous system (Shields differentiate which specific receptor and post-receptor and Eccleston, 19731, and platelets (Marmaras and second messenger systems mediate the autocrine effect Mimikos, 1971). Among the numerous actions of 5-HT, of 5-HT on growth of BON cells, selective receptor recent studies suggest that 5-HT has a growth-stimu- antagonists, SDZ 21-009 (5-HT,, and 5-HT,, receptor latory activity for Chinese hamster lung fibroblasts antagonist) and mesulergine (CU 32-085 [5-HTIc and (Seuwen et al., 19881, bovine vascular smooth muscle 5-HT, receptor antagonist]) have been used. Phosphaticells (Nemecek et al., 1986), rat renal mesangial cells dylinositol (PI) and CAMPproduction have been mea(Takuwa et al., 19891, and rat jejunal crypt cells (Tut- sured in response to 5-HT or the antagonists. ton and Barkla, 1986). MATERIALS AND METHODS Overproduction of 5-HT is often observed in patients The following substances were purchased: Dulbeco’s with carcinoid tumors (Lembeck, 1953; Thorson et al., 1954; Mazzaferri and O’Dorisio, 1987). The ef- modified Eagle’s medium (DMEM) and F-12K medium fects of 5-HT on several organs, including gastrointes- (Gibco Laboratories, Grand Island, NY); fetal calf setinal tract, have been studied (Jaffe, 1979) in these rum (FCS) (Hyclone Laboratories, Inc., Logan, UT); and cyclic AMP radiopatients. Although 5-HT is mitogenic for selected cell 3H-thymidine, my~-~H-inositol, types in other species, potential autocrine growth- immunoassay (RIA) kit (Amersham Corporation, Arregulatory effects on human carcinoid tumor cells have lington Heights, IL); Dowex AG-1 x 8 chromatography not been studied because of the lack of a n ideal experimental model. We established the functioning human carcinoid cell line (termed BON) in tissue culture from Received March 26, 1991; accepted August 13, 1991. a pancreatic carcinoid tumor and have maintained it in *To whom reprint requestsicorrespondence should be addressed. C 1992 WILEY-LISS. INC
2
ISHIZUKA ET AL
(formate form) (Bio-Rad Laboratories, Richmond, CAI; other chemicals including 5-HT, ascorbic acid, bovine serum albumin (BSA, RIA grade), isobutylmethylxanthine (IBMX), LiC1, pertussis toxin, 8-bromo-cyclic AMP, 8-bromo-cyclic GMP (Sigma Chemicals, St. Louis, MO), and Rp-adenosine-3’,5’-cyclic monophosphothioate (Rp-CAMPS)(BIOLOG, La Jolla, CAI. Selective 5-HT receptor antagonists, SDZ 21-009 and mesulergine, were kindly supplied by Sandoz Pharma Ltd. (Basel, Switzerland). Concentrations of these 5-HT receptor antagonists employed in this study were selected based on antagonistic efficacies for 5-HT receptors which were provided by Sandoz Pharma. Efficacy and selectivity of SDZ 21-009 has been determined by bioassay methods of Schoeffter et al. (1988) and Bouhelal et al. (1988), and efficacy and selectivity of mesulergine has been determined by bioassay methods of Hoyer et al. (1985) and Doyle et al. (1986). Except for 8-bromo-cyclic AMP and IBMX, all reagents were dissolved with double distilled water. 8-bromo-cyclic AMP was dissolved with 0.01 N NaOH and IBMX was dissolved with 0.2 N NaOH. The above solvents were used appropriately as control vehicles in the present experiments. All experiments were performed using six dishes or wells each time and mean c SEM of a t least three different experiments were shown.
BON cell culture BON cells have been maintained in DMEM and F-12K (1:l) media, containing 5% FCS in a humidified atmosphere of 95% air and 5% COz at 37°C. Stock cultures have media changed every three days (doubling time is approximately 48 hours). In order to compare results between experiments, BON cells in passages 9-13 were utilized in the present study. Effect of 5-HT and 5-HT receptor antagonists on 3H-thymidineincorporation BON cells (3 x lo5) grown in DMEM and F-12K (1:l) containing 5% FCS were rendered quiescent by complete deprivation of serum for 24 hours. Then a DMEMi F-12K containing 3H-thymidine (1Ciiml) and reagents, 5-HT (lo-’ to M) alone or with SDZ 21-009 M) or mesulergine (lop7M), were added. After another 24 hours, cell monolayers were fixed with 5% trichloroacetic acid (TCA) and washed twice. Acid-insoluble material was collected with 0.1 N NaOH and counted by a liquid scintillation spectrometry. Ascorbic acid (0.01%), which prevents degradation of 5-HT, was added to solutions of all reagents, including vehicles for control, in this experiment and following all experiments. Effect of 5-HT and 5-HT receptor antagonists on cell growth BON cells (1 x lo5)grown in DMEM/F-lBK containing 5% FCS had medium replaced by the same culture medium containing 0.5% FCS to examine the stimulatory effect of 5-HT on cell growth. After two days in culture, 5-HT (lop7, and lop5 M), SDZ 21-009 (lo-’ to M), and mesulergine M) were added alone or in combination every other day. Medium was replaced every other day. Every other day, cells to be counted were detached from dishes with trypsin (1:250,
Gibco) and diluted tenfold with Isoton (Curtin Matheson Scientific, Houston, TX) to stop the action of trypsin. Then, cell number was counted by a Coulter counter which was electrically set up to count cells under 100 p,m in size. Cell number as determined by a Coulter counter was similar (less than 5% difference) to viable cell number determined by a dye (trypan blue) exclusion method using a hemocytometer. Also, coefficient variances of low and high counts determined by this electric counter were less than 2%.
Effect of 5-HT and 5-HT receptor antagonists on PI hydrolysis PI hydrolysis (inositol monophosphate formation [IP]) was measured following a protocol adapted from Hawkins et al. (1989). In brief, BON cells (3 x lo5), which were harvested by trypsin (1:250) and cultured in 24-well tissue culture plate for 2 days, were incubated with DMEMIF-12K containing 5% FCS supplemented with my~-~H-inositol (10 p-Ci/ml) for 16 hours. Cells were then incubated for 10 min with oxygenated Krebs-Ringer bicarbonate buffer (KRBB, pH 7.4) containing HEPES (10 mM), BSA (0.1%), glucose (2.5 mM), and LiCl(10 mM). After another 15-min incubation in the presence of 5-HT (lo-’ to lop4 MI alone or with mesulergine (lop7 M) or SDZ 21-009 M), cells were extracted with 10% perchloric acid and immediately neutralized with 6 N KOH. A fraction of I P was extracted by anion exchange chromatography (Dowex AG-1 x 8; formate form; 2 0 0 4 0 0 mesh) using a mixture of formic acid (0.1 M) and ammonium formate (0.2 M) as an eluent. Radioactivity of IP fraction was measured by a liquid scintillation counter. Effect of 5-HT, 5-HT receptor antagonists, or pertussis toxin on the production of cyclic AMP BON cells (5 x lo5),which were harvested by trypsin and cultured in 35 x 10 mm tissue culture dishes for 2 days, were incubated in oxygenated KRBB (pH 7.4) containing HEPES (10 mM), BSA (0.1%), glucose (2.5 mM), and IBMX (1mM). After a 30-min incubation in the presence of 5-HT (lo-’ to M) alone or with SDZ 21-009 (lop7 M) or mesulergine M), cells were extracted with 5% TCA and neutralized with a n excess of CaCO, just before RIA (Tihon et al., 1977). After acetylation of samples with acetic anhydride and triethylamine, intracellular cyclic AMP levels were measured by cyclic AMP RIA kit. To examine the effect of pertussis toxin on cyclic AMP production in BON cells, BON cells were preincubated for 5 hours with or without pertussis toxin (100 ngiml), and then cyclic AMP production in response to 5-HT (lo-’ to M) was examined. Effect of 8-bromo-cyclicAMP or 8-bromo-cyclic GMP on cell growth BON cells (5 x lo4) grown in DMEMIF-12K containing 5%FCS were placed in culture medium containing 0.5% FCS. Forty-eight hours later, 8-bromo-cyclic AMP (lop4M) alone or with 5-HT M), or 8-bromo-cyclic GMP M) alone was added every other day. Culture medium was changed every other day and cell number was counted as described in a previous section.
3
AUTOCRINE EFFECT OF 5-HYDROXYTRYPTAMINE
Effect of Rp-CAMPSon cell growth BON cells (5 x lo4)grown in DMEM/F-lBK containing 5% FCS were placed in culture medium containing 0.5% FCS. Forty-eight hours later, selective cyclic AMP-dependent protein kinase antagonist, Rp-CAMPS (5 x MI, was added every other day. Culture medium was changed every other day and cell number was counted as described in a previous section. Effect of pertussis toxin on 5-HT-stimulated cell growth BON cells (5 x lo4)grown in DMEM/F-l2K containing 5% FCS were placed in culture medium containing 0.5% FCS. Forty-eight hours later, 5-HT (lop5 M) alone, pertussis toxin (100 ng/ml) alone, or 5-HT M) with pertussis toxin (10, 50, and 100 ng/ml) were added every other day. Culture medium was changed every other day and cell number counted. Statistical analysis Data from each study were analyzed by unpaired t-test and were shown as mean 5 SEM of at least three experiments. Significance was assumed for P value less than 0.05.
CI
n
* *
13
I
Effect of 5-HT and 5-HT receptor antagonists on cell growth 5-HT stimulated growth of BON cells in a dosedependent fashion with maximum effect at M (data not shown). The growth-stimulatory effect of 5-HT was completely inhibited by SDZ 21-009 (lop7M), whereas mesulergine had no effect (data not shown). The inhibitory effect of SDZ 21-009 on 5-HT-stimulated growth of BON cells was dose-dependent (data not shown). SDZ 21-009 alone also significantly inhibited the growth of BON cells, while mesulergine alone did not affect the growth of BON cells (Fig. 2). The inhibitory effect of SDZ 21-009 did not persist after cessation of treatment and cell growth recovered rapidly (data not shown). Effect of 5-HT and 5-HT receptor antagonists on PI hydrolysis 5-HT stimulated PI hydrolysis in BON cells in a biphasic dose-dependent fashion and a peak response of PI hydrolysis was observed a t M of 5-HT (Fig. 3). Mesulergine (lop7M) inhibited PI hydrolysis stimulated by 5-HT in a competitive fashion. Thus, the peak response shifted from lop7 M to M of 5-HT. On the other hand, SDZ 21-009 (lop7M) did not affect PI hydrolysis stimulated by 5-HT.
* *
0
c
X
* *
;1 1 0
v
cc I TI -
1 n
9
7
CONTROL
7
8
5
6
4
5-HYDROXYTRYPTAMINE (-LOG M)
Fig. 1. Effects of 5-HT and 5-HT receptor antagonists on 3Hthymidine incorporation of BON cells. In this figure and following figures, error bars indicate standard error of the mean of at least three separate experiments. 0: 5-HT added alone. B: 5-HT with SDZ 21-009 M). * indicatesP < 0.05 vs. M). B: 5-HT with mesulergine control; + indicates P < 0.05 vs. 5-HT alone.
12 -
14
RESULTS Effect of 5-HT and 5-HT receptor antagonists on 3H-thymidineincorporation 5-HT stimulated 3H-thymidine incorporation in serum-deprived BON cells in a dose-dependent fashion / ~ B antagonist, SDZ (Fig. 1).Selective ~ - H T ~ Areceptor 21-009, completely inhibited 3H-thymidine incorporation stimulated by 5-HT, while selective 5-HT,c,z receptor antagonist, mesulergine, failed to affect 3Hthymidine incorporation stimulated by 5-HT.
* *
I2
'z
10
*
-
*
X Y
rK
w
m
3 6
Z
i
w
4
0
2
0
0
2
4
6
DAYS
Fig. 2. Effect of 5-HT and 5-HT receptor antagonists on growth of BON cells. 0: DMEMIF-12 K (1:l)alone. .: 5-HT MI alone. a: SDZ 21-009 M) alone. B:5-HTwith SDZ 21-009. 0: mesulergine M) alone. The day when treatments were started was designated as day 0. * indicates P < 0.05 vs. DMEMIF-12 K alone; + indicates P < 0.05 vs. 5-HT alone.
Effect of 5-HT, 5-HT receptor antagonists, or pertussis toxin on the production of cyclic AMP 5-HT inhibited the production of cyclic AMP in BON cells in a dose-dependent fashion, and SDZ 21-009 Completely prevented the reduction of cyclic AMP production evoked by 5-HT (Fig. 4a). On the other hand, mesulergine did not affect the reduction of cyclic AMP production evoked by 5-HT. SDZ 21-009 alone slightly increased basal CAMPlevel, while mesulergine alone slightly decreased it; however, both effects were not statistically significant (data not shown). Pertussis toxin completely prevented the reduction of cyclic AMP production evoked by 5-HT (Fig. 4b). Per-
ISHIZUKA ET AL
* *1 * *
120
11 :*:
100 -1
2 Iz
00
80
LL 0
Be
60
*
Y
5
4
*
4c CONTROL
a
7 6 5 5-HYDROXYTRYPTAMINE (-LOG M)
4
Fig. 3. Effect of 5-HT alone or with mesulergine or SDZ 21-009 on PI hydrolysis in BON cells. Accumulation of inositol monophosphate (IP) in BON cells was used as a marker of PI hydrolysis. 0:5-HT alone. .: 5-HT with mesulergine M). R : 5-HT with SDZ 21-009 M). Mesulergine or SDZ 21-009 was added 10 min before 5-HT. * indicates P < 0.05 vs. control.
CONTROL
9
8
a
Effect of Rp-CAMPSon cell growth The selective cyclic AMP-dependent protein kinase antagonist, Rp-CAMPS (5 x lop5 MI, significantly stimulated the growth of BON cells (Fig. 7). Effect of pertussis toxin on 5-HT-stimulated cell growth Pertussis toxin alone (100 ng/ml) inhibited the growth of BON cells, and inhibited the growth of BON cells stimulated by 5-HT in a dose-dependent fashion (Fig. 8). The inhibitory effect of pertussis toxin did not persist after cessation of treatment and cell growth recovered rapidly (data not shown). DISCUSSION We have shown in this study that 5-HT is a n autocrine growth factor for human carcinoid tumor cells. Furthermore, the present results indicate that: 1)functioning human pancreatic carcinoid cells have at least two different 5-HT receptors (5-HTIA,,, and 5-HTlc,4; 2) inhibition of cyclic AMP production by 5-HT is mediated through 5-HTlA or 5-HT1, receptors; 3) activation of PI hydrolysis by 5-HT is mediated through 5-HTlc or 5-HT2 receptors; 4) the autocrine growth effect is transmitted by 5-HTIA or 5-HTIB receptors,
6
5-HYDROXYTRYPTAMINE (-LOG M)
1
110, 100
* \ o * 0 ,‘* *
tussis toxin alone slightly increased basal CAMPlevel; however, its effect was not statistically significant (data not shown).
Effect of 8-bromo-cyclicAMP or 8-bromo-cyclic GMP on cell growth 8-bromo-cyclic AMP M) significantly inhibited the growth of BON cells (Fig. 5a), while 8-bromo-cyclic GMP M) did not affect the growth of BON cells (Fig. 5b). Furthermore, 8-bromo-cyclic AMP ( M) prevented the growth-stimulatory effect of 5-HT M) (Fig. 6). The inhibitory effect of 8-bromo-’cyclicAMP did not persist after cessation of treatment and cell growth recovered rapidly (data not shown).
7
* 1
9 ,
CONTROL
b
9
8
7
6
5
4
5-HYDROXYTRYPTAMINE (-LOG M)
Fig. 4. a: Effect of 5-HT alone (0) or with SDZ 21-009 (0) or mesulergine ( A ) on the production of cyclic AMP in BON cells. b Effect of 5-HT alone ( 0 )or with pertussis toxin (PT) (100 wgiml) ( 0 ) on the production of cyclic AMP in BON cells. Results were expressed as a percent of control. * indicates P < 0.05 vs. control.
activation of which decreases cyclic AMP production apparantly through a pertussis toxin-sensitive inhibitory GTP binding protein; and 5) increase of intracellular cyclic AMP level leads to the inhibition of cell growth, while inhibition of cyclic AMP-dependent protein kinase leads to the stimulation of cell growth. As reported previously (Lawrence et al., 19901, BON cells secrete 5-HT. With a short-term incubation study, we have found that BON cells secrete 5-HT, 0.92 5 0.10 ng/ml/30 min (2.38 x lo-’ M) and 2.24 ? 0.12 ng/ml/60 min (5.78 x lo-’ M) in a serum-free condition (Ishizuka et al., 1990), in which BON cells grow very slowly (doubling time, 144 hours). These data may suggest that BON cells secrete and accumulate, probably in a time- and serum-dependent manner, a sufficient amount of 5-HT which stimulates growth of BON cells in a n autocrine fashion. Previously reported growth-stimulatory effects of 5-HT on non-transformed Chinese hamster lung fibroblasts appear to be produced by inhibition of cyclic AMP production, and this effect is mediated by 5-HTlB receptors (Seuwen et al., 1988). Induction of DNA synthesis by 5-HT in bovine aortic smooth muscle cells
AUTOCRINE EFFECT OF 5-HYDROXYTRYPTAMINE
12-
14-
A
-10-
'
v)
I
!n
I
x
5
1210-
8-
X
W
v
5m
I 3
z
6I
J
- 1 4 W
0
2 0
2
0
B
DAYS Fig. 6. Effect of 8-bromo-cyclic AMP on 5-HT-stimulated growth of BON cells. 0 : DMEMIF-12 K (1:l)alone. . :5-HT M) alone. N: 5-HT with 8-bromo-cyclic AMP (10- MI. The day when treatments were started was designated a s day 0. * indicates P < 0.05 vs. DMEMIF-12 K alone; + indicates P < 0.05 vs. 5-HT alone.
* 2
6
4
*
8
DAYS Fig. 5. A Effect of 8-bromo-cyclic AMP on growth of BON cells. 0: DMEMIF-12 K (1:l) alone. t3:8-bromo-cyclic AMP Mj. B: Effect of 8-bromo-cyclic GMP on growth of BON cells. 0: DMEMIF-12 K (1:l) alone. El: 8-bromo-cyclic GMP MI. The day when treatments were started was considered as day 0. * indicates P < 0.05 vs. DMEMIF-12 K alone.
Y a
m
x 3 z W
0
4
.1
*
0
4
6
8
DAYS
also occurs through a pertussis toxin-sensitive pathway, but is not linked to alterations in cyclic AMP levels (Kavanaugh et al., 1988). The findings of our study suggest that the growth-stimulatory effect of 5-HT on BON cells is mediated through mechanisms similar to those described for Chinese hamster lung fibroblasts (Seuwen e t al., 1988). In the present study, we used relatively selective 5-HT receptor antagonists. SDZ 21-009, thus, antagonizes both 5-HTlA and 5-HTlB receptors. Previous reports have suggested that activation of either 5-HTl, or 5-HTlB receptors inhibits the production of cyclic AMP (Schoeffter and Hoyer, 1988; Bouhelal et al., 1988). On the other hand, there is a consensus that in bovine or human cells, 5-HTlB receptor is never present; however, a pharmacologically different but functionally equivalent receptor, 5-HTlD, may be expressed instead of 5-HTlB receptor (Hoyer and Middlemiss, 1989). Furthermore, activation of the 5-HTlD receptor has been shown to inhibit adenylate cyclase (Schoeffter et al., 1988; Hoyer and Scheoffter, 1988). The effects of the membvane-permeable synthetic cyclic AMP analogue, 8-bromo-cyclic AMP, that significantly inhibited growth of BON cells support the conclusion that 5-HT stimulates growth of BON cells by inhibiting the
Fig. 7. Effect of Rp-CAMPSon growth of BON cells. 0 : DMEMIF-12 M). The day when treatment K (1:lj alone. 8 :Rp-CAMPS (5 x with Rp-CAMPS was started was considered a s day 0. * indicates P < 0.05 vs. DMEMIF-12 K alone.
production of cyclic AMP. Taken together, since SDZ 21-009 cannot antagonize 5-HTlD receptor in the concentrations employed in the present study (based on the information provided by Sandoz Pharmaceutical), the inhibitory effect on the production of cyclic AMP in BON cells is most likely mediated through 5-HT1, receptor. The selective cyclic AMP-dependent protein kinase antagonist, Rp-CAMPS,stimulated the growth of BON cells. This antagonist inhibits the activated cyclic AMP-dependent protein kinase (Pereira et al., 1987; Erneux et al., 1986); therefore, the finding that RpCAMPS alone stimulates BON cell growth suggests that cyclic AMP-dependent protein kinase in BON cells is constantly stimulated in tissue culture and maintains a tonic negative control of BON cell growth. High concentrations of cyclic AMP, as used in the
ISHIZUKA ET AL
6 *
5 m '
z
#
ACKNOWLEDGMENTS The authors would like to thank Jell Hsieh and Mary Schmitz-Brown for their technical assistance and Kelly Lee, Sharon Llewellyn, and Cathy Gehret for their help in the preparation of this manuscript. Supported by grants from the National Institutes of Health (5R37 DK 15241, PO1 DK 35608,5Kll CA 01309) and by a grant from the American Cancer Society (PDT-220).
4
x v r Y 3 W
m
I 2
2
2
3 W 0
tients with carcinoid tumor may also inhibit growth of carcinoid tumor cells.
1
LITERATURE CITED 0 0
2
4
DAYS
Fig. 8. Effect of pertussis toxin (PT) on 5-HT-stimulated growth of BON cells. 0 : DMEMIF-12 K (1:l) alone. m: 5-HT M) alone. B:PT (100 ngiml) alone. El:5-HT with PT (100 ngiml). 69: 5-HT with PT (50 ngiml). B: 5-HT with PT (10 ngiml). The day when treatments were started was designated as day 0. * indicates P < 0.05 vs. DMEMIF-12 K alone; # indicates P < 0.05 vs. 5-HT alone.
present study, can activate cyclic GMP-dependent protein kinase (Lincoln and Corbin, 1983), which may have growth-regulatory effects (Corbin et al., 1990; Garg and Hassid, 1989). In the present study, 8-bromocyclic GMP did not show any inhibitory effect on the growth of BON cells. These results support the notion that the cyclic AMP pathway is, at least in part, responsible for growth-regulation in BON cells, but cyclic GMP does not have a similar effect. Pertussis toxin inhibited 5-HT-stimulated growth of BON cells and prevented the inhibition of CAMPproduction evoked by exogenous 5-HT. Since pertussis toxin induces ADP-ribosylation of the inhibitory GTP binding protein leading to its functional uncoupling from adenylate cyclase (Ui, 1984), our results suggest that the 5-HT receptors involved in growth-regulatory mechanism in BON cells are coupled to a pertussis toxin-sensitive inhibitory GTP binding protein. In addition to the observations that both SDZ 21-009 and pertussis toxin inhibited growth of BON cells stimulated by 5-HT, we found that addition of either SDZ 21-009 or pertussis toxin in the absence of exogenous 5-HT resulted in inhibition of BON cell growth. These findings suggest that both agents inhibit the growthstimulatory effect of 5-HT that is endogenously released by BON cells; endogenous 5-HT released from BON cells is a n autocrine growth factor for BON cells. Somatostatin or its analogue has been known to inhibit synthesis or release of 5-HT in patients with carcinoid tumor and also to retard tumor growth in some carcinoid patients (Kvols, 1989). These facts may support our notion that 5-HT is an autocrine growth factor for carcinoid tumor cells. 5-HT may play a n important role in pathophysiological aspects of carcinoid tumor in vivo by endocrine, paracrine, and potentially autocrine actions. Although there have been several reports that 5-HT receptor antagonists can improve clinical symptoms related to overproduction of 5-HT (Anderson et al., 1987; Ahlman et al., 19851, our findings suggest that administration of proper selective 5-HT receptor antagonists to pa-
Ahlman, H., Dahlstrom, A,, Gronstad, K., Tisell, L.E., Oberg, K., Zinner, M.J., and Jaffe, B.M. (1985) The pentagastrin test in the diagnosis of the carcinoid syndrome. Ann. Surg., 201:81-86. Anderson, J.V., Coupe, M.O., Morris, J.A., Hodgson, H.J.F., and Bloom, S.R. (1987) Remission of symptoms in carcinoid syndrome with a new 5-hydroxytryptamine M receptor antagonist. Br. Med. J., 294:1129 (abst.). Bouhelal, R.,Smounya, L., and Bockaert, J . (1988) 5-HTI, receptors are negatively coupled with adenylate cyclase in rat substantia nigra. Eur. J. Pharmacol., 151r189-196. Corbin, J.D., Thomas, M.K., Wolfe, L., Shabb, J.B., Woodford, T.A., and Francis, S.H. (1990) New insights into cGMP action. In: The Biology and Medicine of Signal Transduction. Y. Nishizuka, M. Endo, and C. Tanaka, eds. Raven Press, New York, pp. 411-418. Doyle, V.M., Creba, J.A., Ruegg, U.T., and Hoyer, D. (1986) Serotonin increases the production of inositol phosphates and mobilizes calcium via the 5-HT2 receptor in A7r5 smooth muscle cells. Naunyn Schmiedebergs Arch. Pharmacol., 333:98-103. Erneux, C., Van Sande, J., Jastorff, B., and Dumont, E. (1986) Modulation of cyclic AMP action in the dog thyroid by its agonist and antagonist Sp- and Rp-adenosine 3',5'-monophosphorothioate. Biochem. J.,234:193-197. Garg, U.C., and Hassid, A. (1989) Inhibition of rat mesangial cell mitogenesis by nitric oxidegenerating vasodilators. Am. J . Physiol., 257:F60-F66. Hawkins, D., Enyedi, P., and Brown, E. (1989) The effects of high extracellular Ca2+and Mg2+concentrations on the levels of inositol 1,3,4,5-tetrakisphosphatein bovine parathyroid cells. Endocrinology, 124:838-844. Hoyer, D., Emgel, G., and Kalkman, H.O. (1985) Molecular pharmacology of 5-HT, and 5-HT, recognition sites in rat and pig brain membranes: Radioligand binding studies with L3Hl 5-HT, ["HI 8-OH-DPAT, (-) [lz5I] iodocyanopindolol, r3HI mesulergine and f3HJ ketanserin. Eur. J . Pharmacol., 118:13-23. Hoyer, D., and Middlemiss, D.N. (1989) Species differences in the pharmacology of terminal 5-HT autoreceptors in mammalian brain. TiPS 10:130-132. Hoyer, D., and Scheoffter, P. 11988) 5-HT,, receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in calf substantia nigra. Eur. J. Pharmacol., 147:145-147. Ishizuka, J., Parekh, D., Karp, G.W., Haber, B., Greeley, G.H., Jr., Townsend, C.M., Jr., and Thompson, J.C. (1990) Evidence of a cholinergic receptor on long-term cultured human pancreatic carcinoid cells. Gastroenterology, 98266 (abst.). Jaffe, B.M. (1979) Prostaglandins and serotonin: Nonpeptide diarrheogenic hormones. World J. Surg. 3565-578. Kavanaugh, W.M., Williams, L.T., Ives, H.E., and Coughlin, S.R. (1988) Serotonin-induced deoxyribonucleic acid synthesis in vascular smooth muscle cells involves a novel, pertussis toxin-sensitive pathway. Mol. Endocrinol., 123599-605, Kvols, L.K. (1989) Therapeutic considerations for the malignant carcinoid syndrome. Acta Oncologica, 28:433438. Lawrence, J.P., Ishizuka, J., Harber, B., Townsend, C.M., Jr., and Thompson, J.C. (1990) The effect of somatostatin on 5-hydroxytryptamine release from a carcinoid tumor. Surgery, 108;11311135. Lembeck, F. (1953) 5-Hydroxytryptamine in a carcinoid tumour. Nature, 172t910-911. Lincoln, T.M., and Corbin, J.D. (1983)Characterization and biological role of the cGMP-dependent protein kinase. In: Advances in C&dic Nucleotide Research. P. Greengard and G.A. Robison, eds. Raven Press, New York, Vol. 15, pp. 139-192. Marmaras, V.J., and Mimikos, N. (1971) Enzymic formation of serotonin in mammalian blood platelets and red cells. Experientia, 27,196-197.
AUTOCRINE EFFECT OF 5-HYDROXYTRYPTAMINE
Mazzaferri, E.L., and O’Dorisio, T.M. (1987) Introduction: Endocrine tumors-special problems in diagnosis and management. Semin. Oncol., 14:235-236. Nemecek, G.M., Coughlin, S.R., Handley, D.A., and Moskowitz, M.A. (1986) Stimulation of aortic smooth muscle cell mitogenesis by serotonin. Proc. Natl. Acad. Sci. U.S.A., 83t674-678. Pereira, M.E., Segaloff, D.L., Ascoli, M., and Eckstein, F. (1987) Inhibition of choriogonadotropin-activated steroidogenesis in cultured leydig tumor cells by the Rp diastereoisomer of adenosine 3’3’-cyclic phosphorothioate. J. Biol. Chem., 262t6093-6100. Schoeffter, P., and Hoyer, D. (1988) Centrally acting hypotensive agents with affinity for 5-HT,, binding sites inhibit forskolinstimulated adenylate cyclase activity in calf hippocampus. Br. J. Pharmacol., 95t975-985. Schoeffter, P., Waeber, C., Palacios, J.M., and Hoyer, D. (1988) The 5-hydroxytryptamine5-HTIDreceptor subtype is negatively coupled to adenylate cyclase in calf substantia nigra. Arch. Pharmacol., 337t602-608. Seuwen, K., Magnaldo, I., and Pouyssegur, J. (1988) Serotonin stimulates DNA synthesis in fibroblasts acting through 5-HT,, receptors coupled to a GI-protein.Nature, 335t254-256. Shields, P.J., and Eccleston, D. (1973) Evidence for the synthesis and
7
storage of 5-hydroxytryptamine in two separate pools in the brain. J. Neurochem., 20:881-888. Sjolund, K., Sanden, G., Hakanson, R., and Sundler, F. (1983) Endocrine cells in human intestine: An immunocytochemical study. Gastroenterology, 85:1120-1130. Takuwa, N., Ganz, M., Takuwa, Y ., Sterzel, R.B., and Rasmussen, H. (1989) Studies of the mitogenic effect of serotonin in rat renal mesangial cells. Am. J. Physiol., 257tF431-F439. Thorson, A., Biorck, G., Bjorkman, G., and Waldenstrom, J. (1954) Malignant carcinoid of the small intestine and metastases to the liver, valvular disease of the right heart (pulmonary stenosis and tricuspid regurgitation without septa1 defect),peripheral vasomotor symptoms, bronchoconstriction and unusual type of cyanosis. Am. Heart J., 47:795-817. Tihon, C., Goren, M.B., Spitz, E., and Rickenberg, H.V. (1977) Convenient elimination of trichloroacetic acid prior to radioimmunoassay of cyclic nucleotides. Analyt. Biochem., 80r652-653. Tutton, P.J.M., and Barkla, D.H. (1986)Serotonin receptors influencing cell proliferation in the jejunal crypt epithelium and in colonic adenocarcinomas. Anticancer Res., 6t1123-1126. Ui, M. (1984) Islet activating protein, pertussis toxin: A probe for functions of the inhibitory guanine nucleotide regulatory component of adenylate cyclase. Trends Pharmacol. Sci., 5277-279.
