Life Sciences 71 (2002) 667 – 678
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`
`Expression of dopamine receptors and transporter in
`neuroendocrine gastrointestinal tumor cells
`
`K. Lemmer a, G. Ahnert-Hilger b, M. Ho¨pfner a, S. Hoegerle c, S. Faiss a, P. Grabowski a,
`M. Jockers-Scheru¨bl d, E.O. Riecken a, M. Zeitz a, H. Scheru¨bl a,*
`
`aMed. Klinik I, Universita¨tsklinikum Benjamin Franklin, FU Berlin, Germany
`bInst. Anatomie, HU, Charite´ Mitte, Berlin, Germany
`cAbt. Nuklearmedizin, Albert-Ludwigs Universita¨t, Freiburg, Germany
`dPsych. Klinik, Universita¨tsklinikum Benjamin Franklin, FU Berlin, Germany
`
`Received 17 July 2001; accepted 14 January 2002
`
`Abstract
`
`C-11- or F-18-DOPA positron emission tomography (DOPA PET) is a new sensitive imaging technique for
`small neuroendocrine gastrointestinal tumors which evaluates the decarboxylase activity. To further characterize
`the dopaminergic system in neuroendocrine gastrointestinal tumor cells, we investigated the expression of both
`dopamine receptors and the transmembrane dopamine transporter
`(DAT)
`in the human neuroendocrine
`pancreatic cell line BON and in the neuroendocrine gut cell line STC-1. Both BON and STC-1 cells expressed
`mRNA of the dopamine receptors D2 – D5 and DAT. mRNA of the dopamine receptor D1 was detected in BON
`cells only. Both in BON and STC-1 cells, expression of D2 and D5 receptors and DAT was also demonstrated
`immunocytochemically. For functional receptor characterization intracellular cAMP levels ([cAMP]i) were
`determined. Whereas in STC-1 cells dopamine and the D1-like (D1/D5) receptor agonist SKF 38393 increased
`[cAMP]i, [cAMP]i was decreased by dopamine or the D2-like (D2 – D4) receptor agonist quinpirole in BON
`cells. Functional DAT activity was, however, not detected in either cell line. The presence of both dopamine
`receptors and of the DAT suggests an autocrine and/or paracrine function of dopamine in neuroendocrine
`gastrointestinal tumor cells. Yet neither the transmembrane dopamine transporter nor dopamine receptors are
`likely to contribute to positive DOPA PET imaging of neuroendocrine gastrointestinal tumors. However, these
`
`* Corresponding author. Tel.: +49-30-8445-3534; fax: +49-30-8445-4481.
`E-mail address: hscher@zedat.fu-berlin.de (H. Scheru¨bl).
`
`0024-3205/02/$ - see front matter D 2002 Published by Elsevier Science Inc.
`PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 7 0 3 - 4
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`molecules may be of diagnostic importance when applying other dopaminergic system tracers. D 2002
`Published by Elsevier Science Inc.
`
`Keywords: DOPA PET; Dopamine transporter; Dopamine receptor; Carcinoid; cAMP
`
`Introduction
`
`Neuroendocrine gastrointestinal tumors, i.e. carcinoids and endocrine pancreatic tumors, are often
`small and difficult to localize. Recently, C-11- or F-18-DOPA positron emission tomography (DOPA
`PET) has been introduced as a new sensitive imaging technique for the detection and staging of these
`tumors [1,2]. In view of the possibilities of PET to study metabolic changes (during therapy), its future
`clinical application may well extend beyond diagnostic use.
`Gastrointestinal neuroendocrine tumors belong to the amine precursor uptake and decarboxylation
`(APUD) cell system and have the capacity to uptake and to decarboxylate amine precursors such as 5-
`hydroxytryptophan. The re-uptake of monoamines most likely proceeds via selective sodium/chloride
`dependent monoamine transporters of the plasma membrane [3,4]. Accumulation of monoamines into
`secretory vesicles is achieved by vesicular monoamine transporters [5,6]. Dopa is readily decarboxylated
`to dopamine in peripheral tissues [7] and the decarboxylase activity is thought to be essential for positive
`DOPA PET imaging. Although dopamine, its receptors and the transmembrane dopamine transporter
`(DAT) are known to play an important role in gastrointestinal physiology [8 –11], they have not been
`investigated in neuroendocrine gastrointestinal tumor cells so far.
`Dopamine receptors belong to the family of G protein-coupled receptors. They modulate the activity
`of adenylate cyclases (AC) via G proteins. Based on their AC interactions the five known dopamine
`receptors are divided into two subfamilies, i.e. the D1-like (D1 and D5) and the D2-like (D2, D3 and D4)
`receptors. D1-like receptors stimulate AC activity via Gs proteins, while D2-like receptors decrease
`intracellular cAMP accumulation ([cAMP]i) via Gi/Go proteins [12,13]. As for the transmembrane
`dopamine transporter different subtypes have not been described so far [3,14].
`In this study we investigated the expression of the dopamine receptors D1– D5 and the trans-
`membrane dopamine transporter DAT in the human pancreatic neuroendocrine tumor cell line BON and
`in the neuroendocrine gut tumor cell line STC-1. In addition to mRNA and immunofluorescence studies,
`we functionally characterized dopamine receptors and the transmembrane dopamine transporter.
`
`Material and methods
`
`Cell cultures
`
`STC-1 cells, derived from a murine neuroendocrine gut tumor, were cultured in Dulbecco’s modified
`Eagle medium (DMEM) supplemented with 15% horse serum, 2.5% fetal calf serum (FCS) and 1% L-
`glutamine at 37 jC in a 5% CO2 humidified atmosphere [17]. The human pancreatic neuroendocrine
`(carcinoid) cell line BON was maintained in a mixture of DMEM and F12K medium (1:1) supplemented
`with 10% FCS and 1% L-glutamine. BON cells were cultured at 37 jC in a 5% CO2 humidified
`atmosphere [15].
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`Reverse transcription-polymerase chain reaction (RT-PCR)
`
`RNAs from both cell lines, and from mouse and human brain (cortex) tissues, were isolated with a
`commercial RNA isolation kit (PURE script, Biozym, Oldendorf, Germany). To eliminate residual
`genomic DNA, the RNA samples were treated with DNase I (Gibco, Eggenstein, Germany) for 15 min at
`room temperature. Single strand cDNA was generated from the cellular mRNA by reverse transcription
`using Superscript II RNase H Reverse Transcriptase (Gibco) as described previously [16]. Reverse
`transcription and amplification conditions were carried out as described previously [17]. The oligonu-
`cleotide primers (Amersham Pharmacia, Freiburg, Germany) used for amplification of the dopamine
`receptor subtypes D1–D5 and DAT (Table 1) were designed from GenBank sequences with BLASTN
`2.0 software, NCBI (National Center for Biotechnology Information, Bethesda, MD, USA) or selected
`from published primer sequences [18– 23]. PCR started with an inital denaturation step for 5 min at 94
`jC. Maintaining the samples at 85 jC 1.25 U Taq polymerase (Promega, Mannheim, Germany) was
`added. Cyclic denaturation at 94 jC for 45 s and elongation at 72 jC for 2 min were identical in each
`PCR, with different annealing temperatures for respective primers as shown in Table 1. Amplification
`consisted of 38 cycles. PCR products were visualized in ethidium bromide-stained 1.8% agarose gels
`and band lengths were compared with a 123 bp DNA ladder (Gibco).
`
`Table 1
`RT-PCR primers used for the detection of dopamine receptors D1 – D5 or the transmembrane dopamine transporter DAT
`
`Subtype Species Primer sequence
`
`D1
`
`mouse
`
`human
`
`human/
`mouse
`human
`
`D2
`
`D2S
`
`D3
`
`mouse
`
`human
`
`D4
`
`mouse
`
`human
`
`D5
`
`mouse
`
`human
`
`DAT
`
`mouse
`
`human
`
`5V-GTAGCCATTATGATCGTCAC-3V
`5V-GATCACAGACAGTGTCTTCAG-3V
`5V-CAGTCCACGCCAAGAATTGCC-3V
`5V-ATTGCACTCCTTGGAGATGGAGCC-3V
`5V-GCAGCCGAGCTTTCAGGGCC-3V
`5V-GGGATGTTGCAGTCACAGTG-3V
`5V-GAGGGCTCCACTAAAGGAGG-3V
`5V-GGGATGTTGCAGTCACAGTG-3V
`5V-AGGTTTCTGTCAGATGCC-3V
`5V-GTTGCTGAGTTTTCGAACC-3V
`5V-GCGTTACTACAGCATCTGCCAGGAC-3V
`5V-AGACAGGATCTTGAGGAAGG-3V
`5V-CACCAACTACTTCATCGTGA-3V
`5V-AAGGAGCAGACGGACGAGTA-3V
`5V-TGCTGTGCTGGACGCCCTTCTTCG-3V
`5V-CGTTGCGGAACTCGGCGTTGAAGA-3V
`5V-CTACGAGCGCAAGATGACC-3V
`5V-CTCTGAGCATGCTCAGCTG-3V
`5V-GTCGCCGAGGTGGCCGGTTAC-3V
`5V-GCTGGAGTCACAGTTCTCTGCAT-3V
`5V-GGCTTACAGGACCTCAAAG-3V
`5V-TGAACCTCCACTGGTGTCT-3V
`5V-AGCAGAACGGAGTGCAGCT-3V
`5V-GTATGCTCTGATGCCGTCT-3
`
`Annealing
`temperature
`55 jC
`
`RT-PCR
`product
`
`213 bp
`
`GenBank accession
`number/Reference
`
`Acc. no. L20336
`
`60 jC
`
`60 jC
`
`60 jC
`
`55 jC
`
`55 jC
`
`58 jC
`
`55 jC
`
`58 jC
`
`60 jC
`
`58 jC
`
`55 jC
`
`455 bp
`
`529 bp (D2L)
`442 bp (D2S)
`417 bp
`
`Vrana et al. 1995 [20]
`Acc. no. X55760
`Vrana et al. 1995 [20]
`Acc. no. S69899
`Acc. no. S69899
`
`276 bp
`
`Fishburn et al. 1993 [18]
`
`439 bp
`
`Segal et al. 1997 [23]
`
`393 bp
`
`162 bp
`
`359 bp
`
`363 bp
`
`258 bp
`
`Matsumoto et al. 1995 [19]
`Acc. no. U19880
`Mulcrone and Kerwin
`1996 [21]
`Acc. no. L20330
`
`Nagai et al. 1996 [22]
`Acc. no. X58454
`Acc. no. U16265
`
`785 bp
`
`Acc. no. L24178
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`Immunocytochemistry
`
`STC-1 and BON cells were grown on 12 mm coverslips for 3–4 days. The cells were gently washed 3
`times for 5 minutes with icecold 0.15 M PBS (pH 7.4) and fixed with 3.5% formaline dissolved in PBS
`for 20 min at room temperature. Membrane permeabilization was achieved by incubating the cells with
`0.2% Triton X-100 for 10 min. Nonspecific binding was blocked with 1% BSA in PBS for 1 h. The cells
`were then incubated for 1 h with polyclonal antisera (1 Ag IgG/ml) diluted with 0.3% BSA in PBS. The
`specific antibodies had been raised against amino- or carboxy-terminal peptides of the D2 receptor (N-
`19; Santa Cruz Biotechnology, Heidelberg, Germany), the D5 receptor (R-18; C-20) or DAT (N-19).
`Each coverslip was overlaid with 100 Al antibody solution. Negative controls were prepared by
`incubation with polyclonal antibodies that had been neutralized by a five fold excess of their antigens
`(blocking peptides; Santa Cruz Biotechnology). Immunocomplexes were detected with fluorescein-
`conjugated secondary antibodies (Santa Cruz Biotechnology). The rinsed coverslips were dried, mounted
`on slides with Aquamount (Polysciences, St. Goar, Germany) and examined on an Axioskop-2
`microscope (Zeiss, Jena, Germany).
`
`cAMP assay
`
`cAMP accumulation was determined with a competitive enzyme immunoassay (BiotrakTM; Amer-
`sham Pharmacia Biotech, Braunschweig, Germany). STC-1 and BON cells were seeded at a concen-
`tration of 50,000 cells/well in 96 well microtitre plates and cultured for 48 h. The cells were washed
`twice with sodium bath solution containing 130 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 10 mM glucose
`and 10 mM Hepes, pH 7.3 (37 jC). Cells were then incubated in 200 Al sodium bath solution
`supplemented with 100 AM isobutylmethylxanthine (IBMX) (Sigma, Deisenhofen, Germany) in the
`absence or presence of 1 AM forskolin (Sigma) and/or the following drugs: 100 AM dopamine, 100 AM
`SKF 38393 or 100 AM quinpirole (Sigma). Incubation was carried out for 15 min at 37 jC. Following
`two rinses with sodium bath solution 200 Al lysis reagent was added. Cells were agitated for up to 15
`min and cell lysis was verified by microscopic evaluation. 100 Al aliquot of cell lysate were transfered to
`the 96 well microtitre plate and the assay was carried out as described previously [24]. The cAMP values
`(fmol/well) of the samples were calculated by direct read-off from the standard curve.
`
`[3H] dopamine uptake
`
`Primary neuronal cultures were prepared from embryonic (ED 15) mouse mesencephalon and
`cultured for 2 weeks as described previously [25]. BON and STC-1 cells were grown for 48 h. Cells
`were preloaded with [3H] dopamine in serum free medium supplemented with 1 mM ascorbic acid in the
`absence or presence of 0.1–1 AM GBR 12783, a selective DAT inhibitor. Uptake of [3H] dopamine was
`stopped by several washes after 4 h of incubation at 37 jC. Radioactivity as well as protein content were
`estimated after lysing the cells in 0.1% Triton X-100.
`
`Statistics
`
`Results are expressed as means F standard error. Data were analyzed using Student’s non-paired two-
`tailed t-test.
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`Results and discussion
`
`Expression of dopamine receptor and DAT mRNAs
`
`RT-PCR revealed mRNA expression of dopamine receptors and DAT in STC-1 and BON cells (Fig.
`1). While STC-1 cells expressed mRNA of D2–D5 receptors only, BON cells expressed mRNA of all
`five dopamine receptors. In the two cell lines mRNA of both the long and the short D2 isoforms, D2L
`and D2S, generated by alternative splicing [26] were present. In STC-1 cells, D2L mRNA amplification
`resulted in an abundant band, whereas D2S mRNA showed a weaker amplification, which suggests a
`predominance of the longer D2 isoform in STC-1 cells [20]. In BON cells, mRNA amplification with the
`primer pair D2 resulted in an abundant D2L band but in contrast to brain no D2S amplification could be
`observed. By use of a D2S mRNA selective forward primer (D2S primer pair listed in Table 1) mRNA
`amplification of BON cells resulted in a marked D2S band. These findings suggest that D2S mRNA
`amplification with the primer pair D2 was competitively inhibited by large amounts of D2L mRNA.
`
`Fig. 1. Agarose gel electrophoresis with PCR products of dopamine receptors and the dopamine transporter in STC-1 and
`BON cells. Top panel: Amplification was performed with cDNA from mouse brain as control (C) and from STC-1 cells (S).
`The primers used corresponded to the dopamine receptors D1, D2 (amplifying both the long and short isoforms D2L and
`D2S), D3, D4 or D5 and to the transmembrane dopamine transporter DAT. M, 123 bp DNA ladder. Bottom panel:
`Amplification was performed with cDNA from human brain as control (C) and from BON cells (B). The primers used
`corresponded to the dopamine receptors D1, D2L/S, D2S, D3, D4 or D5 and to the transmembrane dopamine transporter DAT.
`M, 123 bp DNA ladder.
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`Thus, STC-1 cells as well as (mouse and human) brain seem to express lower amounts of D2L mRNA in
`relation to D2S mRNA than BON cells do. Although both D3 and D4 mRNAs are rarely found in the
`gastrointestinal tract of the rat [9,11], both D3 and D4 mRNAs were expressed in the human BON and in
`the murine STC-1 cells. In STC-1 cells only a D3 mRNA band of 276 bp length was obtained (D3L),
`while the shorter splice variant of 213 bp length [18] was not observed. Human D3 mRNA was
`amplified in low amount in BON cells, resulting in a single band equivalent in length to D3L mRNA.
`Both cell lines expressed mRNA of the D5 receptor which has been described as the most prevalent
`dopamine receptor in the upper gastrointestinal tract [27–30]. The expression patterns of dopamine
`receptor mRNAs in STC-1 and BON cells were reproducible with cells of different passages. Thus,
`similar to the situation in neurons [20], STC-1 as well as BON cells co-express mRNAs of both
`subfamilies of dopamine receptors, i.e. the [cAMP]i increasing D1-like (D1, D5) receptors and the
`[cAMP]i decreasing D2-like (D2–D4) receptors. While DAT mRNA was persistently expressed in STC-
`1 cells, BON cells did not express DAT mRNA stably in different cell passages.
`
`Immunocytochemical studies of dopamine receptors and DAT
`
`D2 and D5 receptors are known to be widely distributed in the upper gastrointestinal tract [11,29].
`The antibodies used specifically recognize epitops of the extracellular N terminus or cytoplasmic
`C-terminus of the receptors or the intracellularly localized N-terminus of DAT. To access cytoplasmic
`targets,
`the cell membranes were permeabilized by incubation with 0.2% Triton X-100. Fig. 2
`demonstrates the immunoreactivity of STC-1 and BON cells after incubation with polyclonal antibodies
`against the D2 receptor (Fig. 2A and 2E) and the D5 receptor (Fig. 2C and 2G).
`Both cell lines were also immunopositive for DAT (Fig. 3A and 3C). But in contrast to the STC-1 cell
`line only about 25% of BON cells showed a bright fluorescence for DAT (in three independent
`experiments). This may correspond to the observed unstable expression of DAT mRNA or the diversity
`of BON cells. The immunofluorescence signals of DAT, D2 or D5 receptors were interspersed
`homogeneously only sparing nuclei. Preabsorption of the antibodies with immunizing peptides
`eliminated the strong immunofluorescences (Figs. 2B, 2D, 2F, 2H, 3B and 3D). Hence, both STC-1
`and BON cells do co-express D2 and D5 receptors as well as DAT. Interestingly D2, D5 and DAT
`immunoreactivity had been described previously in the gastrointestinal tract. Thus, gastric and duodenal
`mucosa and pancreatic cells express DAT together with tyrosine hydroxylase activity, high levels of
`dopamine, vesicular monoamine transporters (VMAT) and D5 mRNA [9,11,27,29,30]. Similarly,
`VMAT1 and VMAT2 expression could be detected immunocytochemically in BON cells [6].
`
`Adenylate cyclase activity and dopamine receptors
`
`To study dopamine receptor function, adenylate cyclase (AC) activity both of untreated and of
`forskolin-stimulated STC-1 and BON cells was investigated in the absence or presence of either
`dopamine, the D1-like receptor agonist SKF 38393 or the D2-like receptor agonist quinpirole. To block
`phosphodiesterase activity, cells were treated with 1 AM IBMX. In unstimulated STC-1 cells 100 AM
`dopamine as well as 100 AM SKF 38393 increased basal [cAMP]i by about 35% (p < 0.05) (Fig. 4).
`Incubation with 1 AM forskolin, a direct activator of the catalytic subunit of AC resulted in about a two
`fold increase of [cAMP]i (p < 0.05). Co-incubation with dopamine or SKF 38393 potentiated the
`forskolin-stimulated cAMP accumulation by about 230% and 150%, respectively (p < 0.05). The
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`Fig. 2. Expression of D2 and D5 dopamine receptors in STC-1 (A – D) and in BON cells (E – H). Cells were fixed,
`permeabilized and immunostained as described in materials and methods. Immunofluorescence microscopy was performed
`using a conventional fluorescence microscope. STC-1 cells were incubated with the D2-specific polyclonal antibody N-19 (A)
`or the D5-specific (mouse reactive) polyclonal antibody R-18 (C) and for control with neutralized antibodies (B, D). Similarly,
`BON cells were incubated with the D2-specific antibody N-19 (E) or the D5-specific (human reactive) antibody C-20 (G) and
`for control with neutralized antibodies (F, H). The immunocomplexes were detected with fluorescein-conjugated secondary
`antibodies. Magnification: 630.
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`Fig. 3. Expression of the transmembrane dopamine transporter DAT in STC-1 (A, B) and in BON cells (C, D). Cells were fixed,
`permeabilized and immunostained as described in materials and methods. Immunofluorescence microscopy was performed
`using a conventional fluorescence microscope. STC-1 (A) and BON cells (C) were incubated with the DAT-specific polyclonal
`antibody N-19 or for control with the neutralized antibody (B, D). The immunocomplexes were detected with a fluorescein-
`conjugated secondary antibody. Magnification 400 (A, B), 630 (C, D).
`
`selective D2 receptor agonist quinpirole did not decrease cAMP accumulation in STC-1 cells (data not
`shown).
`In contrast to the stimulatory effect of dopamine in STC-1 cells, dopamine decreased cAMP
`accumulation in BON cells (Fig. 5). In unstimulated BON cells 100 AM dopamine reduced basal
`[cAMP]i by about 40% (p < 0.05). Similar results were obtained with 100 AM quinpirole (p < 0.05).
`Forskolin (1 AM) enhanced [cAMP]i accumulation in BON cells by about 7 fold (p < 0.05). Co-
`incubation with 100 AM dopamine or 100 AM quinpirole decreased the (1 AM) forskolin-induced
`[cAMP]i by about 30% and 20%, respectively (n = 6, p < 0.05). The D1-like receptor agonist SKF 38393
`did not increase either basal or forskolin-induced cAMP accumulation in BON cells (data not shown).
`These results suggest that in BON cells functional D2-like receptors prevail, while in STC-1 cells
`functional D1-like receptors do so.
`
`[ 3H] dopamine uptake
`
`DAT activity in both BON and STC-1 cells was assessed by measurement of [3H] dopamine uptake.
`For comparison [3H] dopamine uptake was also studied in mouse mesencephalon [25] which is known to
`contain dopaminergic neurons from substantia nigra. Intracellular dopamine concentrations differed
`significantly in mesencephalic neurons in the absence or presence of 1 AM of the selective DAT inhibitor
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`Fig. 4. Effects of dopamine (A) and SKF 38393 (B) on the intracellular cAMP level of STC-1 cells. Basal and (1 AM) forskolin-
`stimulated cAMP accumulation were measured in the absence or presence of 100 AM dopamine or 100 AM SKF 38393. Means
`F standard errors (n = 6) are given. *, p < 0.05.
`
`GBR 12783 (9.5 F 1.9 versus 2.7 F 1.1 pmol [3H] dopamine/mg protein, n = 10). In contrast, [3H]
`dopamine uptake was not affected significantly by the DAT inhibitor GBR 12783 (1 AM) neither in BON
`cells (0.13 F 0.01 versus 0.19 F 0.05 pmol/mg protein, n = 10) nor in STC-1 cells (1.5 F 0.9 versus
`0.85 F 0.13 pmol/mg protein; n = 10). Thus, functional DAT activity could not be detected in either
`BON or STC-1 cells.
`The results of this study provide hitherto undescribed evidence for the presence of both dopamine
`receptors and the transmembrane dopamine transporter in neuroendocrine gastrointestinal tumor cells.
`As neuroendocrine tumor cells have the capacity for uptake and decarboxylation of amine precursors
`such as 5-hydroxytryptophan and L-dopa and as they can secrete serotonin and dopamine [31–33], the
`presence of dopamine receptors and of the DAT suggests an autocrine and/or paracrine function of
`dopamine in neuroendocrine gastrointestinal tumor cells.
`Due to the relatively low affinity (high off-rate constant) and competition with relatively high levels
`of endogenous dopamine the binding of F-18-dopamine formed from F-18-DOPA to either DAT or
`dopamine receptors will not produce interpretable PET images. Thus, neither the transmembrane
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`Fig. 5. Effects of dopamine (A) and quinpirole (B) on the intracellular cAMP level of BON cells. Basal and (1 AM) forskolin-
`stimulated cAMP accumulation were measured in the absence or presence of 100 AM dopamine or 100 AM quinpirole. Means
`F standard errors (n = 6) are given. *, p < 0.05.
`
`dopamine transporter nor dopamine receptors are likely to contribute to positive DOPA PET imaging of
`neuroendocrine gastrointestinal tumors. However, these molecules may be of diagnostic importance
`when applying other dopaminergic system tracers [34,35].
`
`Acknowledgements
`
`The study was supported by the Deutsche Forschungsgemeinschaft, the Deutsche Krebshilfe and the
`Sonnenfeld-Stiftung. We are indebted to the Institute of Physiology, FU Berlin for lab facilities.
`
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