GASTROENTEROLOGY 1998;114:791–797
`
`Guanylin Stimulates Regulated Secretion From Human
`Neuroendocrine Pancreatic Cells
`
`MATHIAS JOHN,* BERTRAM WIEDENMANN,‡ MOGENS KRUHØFFER,§ KNUT ADERMANN,§
`IEVA ANKORINA–STARK,\ EBERHARD SCHLATTER,\ GUDRUN AHNERT–HILGER,¶
`WOLF–GEORG FORSSMANN,§ and MICHAELA KUHN§
`*Department of Gastroenterology, Klinikum Benjamin Franklin, Freie Universita¨t Berlin, Berlin; §Lower Saxony Institute for Peptide Research,
`Hannover; \Experimentelle Nephrologie, Medizinische Poliklinik, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Mu¨nster; and ‡Universita¨tsklinikum
`Charite´ der Humboldt-Universita¨t zu Berlin, Medizinische Klinik m. S. Hepatologie und Gastroenterologie, Campus Virchow-Klinikum, and
`¶Charite´, Institut fu¨r Anatomie, Humboldt Universita¨t Berlin, Berlin, Germany
`
`Background & Aims: Gastroenteropancreatic neuroen-
`docrine cells secrete chemical messengers in a calcium-
`dependent fashion. So far, other second messenger
`systems involved in regulated secretion have gained
`little attention. The aim of this study was to character-
`ize guanosine 38,58-cyclic monophosphate (cGMP)-
`mediated vesicular secretion in pancreatic neuroendo-
`crine cells. Methods: In a human pancreatic cell line,
`BON, cyclic nucleotide levels and chromogranin A
`release were monitored with specific immunoassays.
`Uptake and release of ␥-aminobutyric acid were mea-
`sured. Intracellular Ca2⫹ concentration was monitored
`with fura-2. Guanylyl cyclase C was analyzed by reverse-
`transcription polymerase chain reaction. Results: Gua-
`nylin increased cGMP concentrations in BON cells via
`guanylyl cyclase C. Stimulation of the cGMP pathway
`by guanylin or Escherichia coli heat-stable enterotoxin
`increased the release of chromogranin A and ␥-amino-
`butyric acid from BON cells. This effect was mimicked
`by the cGMP analogue 8-bromo-cGMP. Conclusions:
`Guanylin and STa stimulate the regulated secretion
`from BON cells via guanylyl cyclase C and cGMP. Our
`study yields novel information about secretory proper-
`ties of guanylin, mediated via a signal transduction
`pathway, increasing cGMP and leading to regulated
`secretion of neuroendocrine cells.
`
`In neuroendocrine cells, two separate, regulated secre-
`
`tory pathways are activated by an elevation of the
`intracellular Ca2⫹ concentration.1,2 Secretory granules/
`large dense core vesicles mediate the release of biogenic
`amines and peptides, and small synaptic vesicle analogues
`store and release amino acid transmitters such as ␥-
`aminobutyric acid (GABA).1,2 The second messenger
`guanosine 38,58-cyclic monophosphate (cGMP) has gained
`increasing attention as an important component in the
`control of widespread cellular functions. In the mamma-
`lian digestive tract, at least three different guanylyl
`cyclases (GCs) are expressed: (1) a receptor GC stimulated
`
`by atrial natriuretic peptide (GC-A), (2) a soluble GC
`stimulated by nitric oxide (NO), and (3) a particulate
`(intestinal) form of receptor GC (GC-C).3
`GC-C, mainly localized in the apical membrane of
`intestinal epithelial cells,
`is activated by heat-stable
`enterotoxins (STa) of several bacteria (i.e., Escherichia coli)
`causing acute secretory diarrhea by enhanced intestinal
`chloride and water secretion.4 Two intestinal peptide
`ligands, guanylin and uroguanylin, support the physi-
`ological significance of GC-C (for review, see Forte and
`Currie5). Because STa and guanylins increase chloride
`secretion across the intestinal mucosa in vitro by elevat-
`ing cGMP6 at the luminal (apical) site of enterocytes, it
`was suggested that these peptides mediate intestinal
`epithelial transport via paracrine mechanisms.
`Little information exists about the effects of cGMP on
`regulated secretory vesicles of neuroendocrine cells of the
`gastroenteropancreatic system. Guanylin immunoreactiv-
`ity has been observed in enterocytes and in endocrine
`pancreas.7 Furthermore, STa-altered cGMP levels in RIN
`1046-38 rat insulinoma cells suggest the expression of
`GC-C in these cells.8 In chromaffin cells of the adrenal
`medulla, guanylin was found to be colocalized with
`catecholamines.9 Thus, the guanylin peptide family could
`play a role in modulating regulated secretion of various
`gastroenteropancreatic neuroendocrine tissues via para-
`crine and autocrine mechanisms.
`The pancreatic cell line BON, derived from a human
`pancreatic neuroendocrine tumor, is a widely used model
`system for neuroendocrine secretion.2,10,11 BON cells
`release serotonin and chromogranin A. The release is
`stimulated by an increase in the intracellular concentra-
`
`Abbreviations used in this paper: 8-Br-cGMP, 8-bromocyclic guano-
`sine monophosphate; DEANO, 1,1-diethyl-2-hydroxy-2-nitroso-
`hydrazine; GABA, ␥-aminobutyric acid; GC, guanylyl cyclase; IBMX,
`38-isobutyl-1-methylxanthine; PCR, polymerase chain reaction; RT,
`reverse transcription; STa, heat-stable enterotoxin.
`r 1998 by the American Gastroenterological Association
`0016-5085/98/$3.00
`
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`792 JOHN ET AL.
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`GASTROENTEROLOGY Vol. 114, No. 4
`
`tion at Ca2⫹ ([Ca2⫹]i) or adenosine 38,58-cyclic monophos-
`phate (cAMP).11 BON cells take up GABA by specific
`transporters and release it in a Ca2⫹-dependent fashion.2
`We have investigated the expression of the guanylin
`receptor, GC-C, compared with other GCs in BON cells.
`The effects of cGMP on secretion of GABA and chromo-
`granin A, secretory marker molecules for both the small
`synaptic vesicle and the large dense core vesicle secretory
`pathway, respectively, were analyzed. We also performed
`fura-2 measurements of [Ca2⫹]i to characterize the events
`leading from cGMP alterations to altered secretion.
`
`Materials and Methods
`Atrial natriuretic peptide (CDD/ANP-99-126) and
`human guanylin were synthesized by Dr. K. Adermann, Lower
`Saxony Institute for Peptide Research (Hannover, Germany).
`Brain natriuretic peptide and C-type natriuretic peptide were
`obtained from Saxon Biochemicals (Hannover, Germany). The
`NO donor 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine (DEANO)
`was from Alexis (Gru¨ nberg, Germany), and BayK 8644 was
`obtained from ICN (Eschwege, Germany). ␥-Amino[2,3-3H]-
`butyric acid ([3H]GABA, 80 Ci/mmol) was purchased from
`Amersham (Braunschweig, Germany). Chromogranin A en-
`zyme-linked immunosorbent assay kits were from DAKO
`(Hamburg, Germany). Fura-2/AM was from Molecular Probes
`(Eugene, OR). All other substances were from Sigma (Deisen-
`hofen, Germany). Except for DEANO, all agents were dis-
`solved in distilled water. DEANO was dissolved with 1
`mmol/L NaOH, and A23187 was dissolved in dimethyl
`sulfoxide. Final concentrations of dimethyl sulfoxide did not
`exceed 0.1% and were without effect in the experiments.
`
`Cell Culture
`BON cells, a gift from C. M. Townsend (Galveston,
`TX) were cultivated as described.2,10 Experiments were per-
`formed with BON cells of passages 36–48, cultured in 24-well
`(⬃5 ⫻ 105 cells/well;
`tissue culture plates
`for 2 days
`⬃125.8 ⫾ 1.8 µg protein/well; mean ⫾ SEM). All experiments
`were performed using three wells each time, and mean ⫾ SEM
`of at least three different experiments are shown.
`
`Measurement of Intracellular Cyclic
`Nucleotide Content
`BON cells were washed twice with oxygenated Krebs-
`Ringer-HEPES buffer (pH 7.4) containing the following
`(mmol/L): NaCl, 130; KCl, 4.7; MgSO4, 1.2; CaCl2, 2.5;
`HEPES, 10; glucose, 11; and 0.2% bovine serum albumin.
`Cells were preincubated at 37°C for 15 minutes in 0.5 mL
`Krebs-Ringer-HEPES buffer with or without 38-isobutyl-1-
`methylxanthine (IBMX, 1 mmol/L). Test agents were added to
`the wells for an additional 30 minutes. Incubation was stopped
`by aspirating the medium, and 0.1 mL ice-cold ethanol (70%,
`vol/vol) was added per well to extract of intracellular cyclic
`nucleotides. After freeze-thawing, extracts were dried by
`
`evaporation and resolved in 1 mL of 50 mmol/L sodium acetate
`buffer (pH 6.0). Solubilized extracts were acetylated, and
`cellular cGMP and cAMP contents were measured with specific
`radioimmunoassays.12,13
`
`Detection of GC-C mRNA
`by Reverse-Transcription Polymerase
`Chain Reaction
`Total RNA was isolated from BON and T84 cells with
`RNeasy (Quiagen, Hilden, Germany). First-strand complemen-
`tary DNA was synthesized from 5 µg RNA as described
`previously.14 One percent of the reverse transcription (RT)
`reaction mixture was mixed with polymerase chain reaction
`(PCR) buffer containing 20 nmol of each deoxynucleoside
`triphosphate and 20 pmol of each primer. The amplification
`was performed in a Perkin-Elmer Cetus thermocycler (Perkin
`Elmer, Stuttgart, Germany) using the following parameters: 5
`minutes of denaturation at 96°C, addition of 1 U Taq
`DNA-polymerase (Biomol, Hamburg, Germany) at 62°C (hot
`start), and 90 seconds of primer extension at 72°C. The
`parameters for the following 34 cycles were denaturation, 96°C
`for 20 seconds; annealing, 62°C for 10 seconds; and extension,
`72°C for 90 seconds. Primers used for PCR amplification of
`GC-C were 58-AGKCAGAASTGCCACAATGGCA (bases 77–
`98), and 58-AAGTACATCTRCTTYCTRGCTGGA (comple-
`mentary to bases 469–492). Negative controls included ampli-
`fication of an RT reaction from which the reverse transcriptase
`was omitted as well as amplification in the absence of template,
`or one primer. The PCR reaction products were analyzed in
`1.5% ethidium bromide–stained agarose gels. To confirm that
`the PCR products were really GC-C, the products were purified
`and directly sequenced using a dye terminator sequencing kit
`(Perkin Elmer).
`
`Measurements of [Ca2ⴙ]i
`[Ca2⫹]i of BON cells was measured with the Ca2⫹-
`sensitive dye fura-2-AM as described previously.15 BON cells of
`passage 48 were grown on glass coverslips (thickness, 0.2 mm;
`diameter, 30 mm), which were mounted on a perfusion
`chamber fixed on the stage of an inverted microscope and
`superfused with phosphate-buffered saline containing 1.3
`mmol/L calcium gluconate (pH 7.4, 37°C). Loaded BON cells
`were excited at 340, 360, and 380 nm, and fura-2 emission was
`recorded at 500–530 nm. The ratio of the emission after
`excitation at 340 and 380 nm at 10 Hz was calculated.
`Fluorescence at 360 nm was used to judge leakage or bleaching
`of fura-2, loss of cells, or air bubbles in the area of measurement
`during the experiments. Calibration of [Ca2⫹]i was performed
`at the end of each experiment by incubation of the cells with
`the Ca2⫹ ionophore ionomycin (1 µmol/L) in the presence (1.3
`mmol/L) and nominal absence of Ca2⫹ (with 5 mmol/L ethylene
`glycol-bis(␤-aminoethyl ether)-N,N,N8,N8-tetraacetic acid). All
`agonists were dissolved in phosphate buffer before the experi-
`ment and tested for at least 4 minutes.
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`GUANYLIN–STIMULATED NEUROENDOCRINE SECRETION 793
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`Regulated Secretion of GABA
`and Chromogranin A
`Secretion experiments were performed with BON cells
`preloaded for 2 hours at 37°C in Krebs-Ringer-HEPES buffer
`containing 0.5 µCi/mL [3H]GABA, 1 mmol/L aminooxyacetic
`acid, and 1 mmol/L gabaculine (for details, see Ahnert-Hilger
`et al.2,16). Aminooxyacetic acid was added to prevent [3H]GABA
`decomposition, and gabaculine is an inhibitor of endogenous
`GABA synthesis and was used to prevent dilution of stored
`[3H]GABA by endogenously synthesized unlabeled GABA.
`Subsequently, preloaded cells were washed three times with
`Krebs-Ringer-HEPES buffer. Treatment with secretagogues
`was performed for 25 minutes at 37°C in the absence (controls)
`or presence of various agents. The secretion was terminated by
`aspiration of the supernatant. [3H]GABA was determined in
`both the supernatant and cell pellet dissolved in lysis buffer
`containing 130 mmol/L Tris-HCl, 10 mmol/L CaCl2, 75
`mmol/L NaCl, pH 8.0, and 0.4% Triton X-100. In addition,
`chromogranin A concentrations were determined in the super-
`natants of untreated and stimulated BON cells using a
`chromogranin A–specific enzyme-linked immunosorbent assay
`(DAKO Diagnostika, Hamburg, Germany). Release is ex-
`pressed as percent of controls; controls without secretagogue
`were set 100%.
`
`Uptake of GABA Into BON Cells
`GABA uptake into intact cells was performed as
`previously described.2 After two washing steps with Krebs-
`Ringer-HEPES buffer, BON cells were incubated in 200 µL
`Krebs-Ringer-HEPES buffer containing [3H]GABA in the
`presence of 1 mmol/L aminooxyacetic acid and 1 mmol/L
`gabaculine for 30 minutes at 37°C. Specificity of the uptake
`was evaluated by the addition of 2 mmol/L unlabeled GABA to
`the incubation mixture. Other substances were added as
`indicated below. The incubation was stopped by aspiration of
`the supernatant. The supernatant was discarded, and the pellet
`was washed again with Krebs-Ringer-HEPES buffer and
`dissolved in lysis buffer (see above). The cell lysate was used for
`the determination of the cellular GABA content by liquid
`scintillation counting and of the protein concentration accord-
`ing to Bradford.17 GABA uptake is expressed as percentage of
`untreated controls (100% uptake).
`Statistics. Results are expressed as mean values ⫾
`SEM. Statistical differences between mean values were deter-
`mined by t test using the statistical functions of GraphPad’s
`Prism (version 2.0) software (GraphPad Software, San Diego,
`CA). P values of ⬍0.05 were considered significant.
`
`Results
`Intracellular cGMP Content in BON Cells
`Resting BON cells contained 109 ⫾ 5 fmol/well
`cGMP. Addition of 1 mmol/L IBMX increased basal
`cGMP levels (240 ⫾ 15 fmol/well; n ⫽ 6). Guanylin
`evoked a concentration-dependent increase in intracellu-
`
`lar cGMP levels, both in the absence and presence of the
`phosphodiesterase inhibitor, with a threshold concentra-
`tion of 10 nmol/L (twofold increment in intracellular
`cGMP). At the maximal guanylin concentration tested (1
`µmol/L, 30 minutes), cGMP levels increased by 54-fold
`without IBMX and by 62-fold in cells that had been
`pretreated with 1 mmol/L IBMX (Figure 1).
`To further characterize the pathways leading to cGMP
`formation in BON cells, the effects of guanylin were
`compared with those of specific activators of other
`membrane-bound receptor guanylyl cyclases. Experi-
`ments were exclusively performed in the presence of
`IBMX. As shown in Figure 2, STa and guanylin stimu-
`lated cGMP formation in BON cells, at 1 µmol/L peptide
`to 85-fold and 61-fold, respectively. The natriuretic
`peptides, atrial natriuretic peptide and brain natriuretic
`peptide, activators of particulate GC-A, also evoked a
`concentration-dependent increase in intracellular cGMP,
`but at a lesser extent (40-fold and 30-fold, respectively, at
`1 µmol/L peptide; Figure 2). In contrast, C-type natri-
`uretic peptide that activates GC-B had no effect on
`cGMP content in BON cells (Figure 2; open triangles).
`The NO donor, DEANO (up to 1 mmol/L) did not
`influence cGMP content in BON cells (Figure 1; control,
`240 ⫾ 60 fmol cGMP/well; DEANO, 1 mmol/L,
`250 ⫾ 50 fmol cGMP/well).
`Intracellular cAMP Content in BON Cells
`Unstimulated BON cells contain 1.2 ⫾ 0.23
`pmol cAMP/well (n ⫽ 6). Upon addition of the adenylyl
`cyclase activator forskolin (10 µmol/L), the phosphodies-
`terase inhibitor, IBMX (1 mmol/L), or the calcium
`ionophore A23187 (5 µmol/L), intracellular cAMP con-
`
`Figure 1. Concentration-dependent effects of guanylin and the NO
`donor DEANO on intracellular cGMP content of BON cells in the
`presence and absence of IBMX (1 mmol/L). Incubation time, 30
`minutes (n ⫽ 4).
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`GASTROENTEROLOGY Vol. 114, No. 4
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`lines, the
`cells (as positive controls). For both cell
`separation of the PCR products by agarose gel electropho-
`resis showed a strong band of the expected size of 439
`base pairs (Figure 4A; GC-C: lane 2, T84; lane 3, BON;
`lanes 4 and 5, negative controls).
`Cytosolic Ca2⫹ Concentration
`The basal fura-2 fluorescence ratio of BON cells
`was 1.28 ⫾ 0.04 (n ⫽ 8). In experiments in which a
`paired calibration at the end of the experiment was
`successful, a corresponding [Ca2⫹]i of 139 ⫾ 18 nmol/L
`was calculated. Addition of guanylin did not influence
`the fura-2 fluorescence ratio (1.14 ⫾ 0.08 at 10 nmol/L
`guanylin and 1.16 ⫾ 0.05 at 0.1 µmol/L guanylin) (see
`also original recording depicted in Figure 5). Consecutive
`additions of acetylcholine (1 µmol/L) and adenosine
`triphosphate (ATP) (100 µmol/L) within the same experi-
`ments (as a functional test of cell viability) induced fast
`and transient increases in the fura-2 fluorescence ratio up
`to 1.95 ⫾ 0.26 (n ⫽ 8) and 1.96 ⫾ 0.33 (n ⫽ 5),
`respectively (Figure 5). Calculated [Ca2⫹]i was raised to
`2.4 ⫾ 1.3 µmol/L by acetylcholine (n ⫽ 7) and to 1.8 ⫾
`1.5 µmol/L by ATP (n ⫽ 5) (peak values). STa, which
`was tested in three separate experiments, did not affect
`the fura-2 fluorescence ratio, i.e., basal [Ca2⫹]i of BON
`cells.
`
`Chromogranin A Secretion in BON Cells
`Basal concentration of chromogranin A released to
`the supernatant from untreated cells (controls) incubated
`for 25 minutes in Krebs-Ringer-HEPES buffer was
`
`Figure 2. Effects of ligands for different membrane-bound guanylyl
`cyclases (GC-A, -B, and -C) on the intracellular cGMP concentration of
`BON cells. Ligands for GC-A (atrial natriuretic peptide, CDD-ANP-99-
`126 [䊐]; brain natriuretic peptide, BNP [䊏]), for GC-B (C-type natri-
`uretic peptide, CNP [䉭]), and for GC-C (guanylin [䊊] and STa [䊉]).
`Basal cGMP concentration of BON cells was 0.23 ⫾ 0.02 pmol/well
`(in the presence of IBMX) (n ⫽ 6–8).
`
`centration increased significantly (Figure 3). The maxi-
`mal effects were elicited by forskolin (6.8-fold increase in
`basal cAMP level). Guanylin, STa, and acetylcholine
`showed no effect (Figure 3).
`BON Cells Express mRNA of GC-C
`To further examine whether the effects of guanylin
`in BON cells were mediated via GC-C, the expression of
`the GC-C was investigated at the mRNA level by
`RT-PCR in BON cells and human colon carcinoma T84
`
`Figure 3. Effects of guanylin, STa, forskolin, IBMX, acetylcholine, and
`A23187 on intracellular cAMP concentration of BON cells. Basal cAMP
`content of BON cells was 1.49 ⫾ 0.2 pmol/well (n ⫽ 6–8). Asterisks
`denote significant effects of the respective substances. P⬍ 0.05 was
`considered significant.
`
`Figure 4. Detection of GC-C mRNA in BON and T84 cells by RT-PCR
`analysis. Amplified DNA was analyzed by electrophoresis on ethidium
`bromide–stained agarose gels (1.5%). A GC-C PCR product of the
`expected size of 416 base pairs was detected in both T84 and BON
`cells (lanes2and 3). The intactness of the complementary DNA was
`tested by amplification of ␤-tubulin (lanes6and 7). No amplification
`products were detected in the respective controls (lanes 4 and 5).
`DNA size markers were pUC18 digested with Sau3AI (lane 1) and
`1-DNA digested with HindIII (lane8).
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`GUANYLIN–STIMULATED NEUROENDOCRINE SECRETION 795
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`GABA Secretion From BON Cells
`Basal GABA release from BON cells was 2.4% ⫾
`0.26% (n ⫽ 13) of the total cellular GABA, determined
`by means of scintillation counting of [3H]GABA. Treat-
`ment with either 50 mmol/L K⫹ or 1 µmol/L BayK 8644
`significantly increased the extracellular GABA concentra-
`tion to 17% above basal level (Figure 7A). In addition,
`regulated secretion of GABA was also enhanced by
`increases in intracellular cGMP. GABA release was
`increased to 16% and 26% above basal level at 0.1 and 1
`µmol/L guanylin, respectively, and to 15% above basal
`level at 1 nmol/L STa (Figure 7A). Again, these effects
`were mimicked by 0.1 mmol/L 8-Br-cGMP (increase of
`20% above basal level) or 0.1 mmol/L dibutyryl-cAMP
`(increase of 38% above basal level). In comparison to
`controls, stimulatory effects were highly significant (P
`values at least ⬍0.05, calculated as given in Materials and
`Methods).
`To exclude that the observed increases in extracellular
`[3H]GABA concentration were due to changes in the
`activity of the plasma membrane transporter instead of an
`exocytotic event by small synaptic vesicle analogues,2
`GABA uptake studies were performed. GABA uptake in
`untreated BON cells (100%) was inhibited by about 87%
`in the presence of 2 mmol/L unlabeled GABA, indicating
`that BON cells possess a specific GABA transport
`system. Neither 1 µmol/L guanylin and 100 µmol/L
`8-Br-cGMP nor 1 µmol/L BayK 8644 inhibited GABA
`uptake (Figure 7B).
`
`Figure 5. Effects of consecutive addition of guanylin, acetylcholine,
`and ATP on the fura-2 fluorescence ratio of BON cells (original
`recording). Measurements were obtained from BON cells grown on
`glass coverslips, passage 48, 3–5 days after seeding.
`
`11.8 ⫾ 4.3 ng/mL (n ⫽ 18). Figure 6A shows the
`secretory effect of guanylin. Chromogranin A release was
`stimulated up to 80% above basal level in the presence of
`1 µmol/L guanylin and to 110% above basal level by STa
`(1 nmol/L). Because IBMX exerted drastic effects on
`chromogranin A secretion (see below), guanylin- and
`STa-stimulated secretion were always analyzed in the
`absence of the phosphodiesterase inhibitor. The secretory
`responses to guanylin and STa were mimicked by the
`membrane-permeable synthetic cGMP analogue 8-Br-
`cGMP (Figure 6A). The effects of substances increasing
`intracellular cAMP (forskolin and IBMX; Figure 6B) or
`Ca2⫹ levels (acetylcholine, A23187, and BayK 8644,
`Figure 6B; potassium depolarization, Figure 6A) are also
`shown in Figure 6. IBMX (1 mmol/L) provided the
`maximal release of chromogranin A of all agents tested
`(nearly 800% above basal level).
`
`Figure 6. Effects of various test agents on the release of chromogranin A by BON cells. (A) Chromogranin A release on potassium depolarization
`and elevation of intracellular cGMP by guanylin, STa, or 8-Br-cGMP. (B) Increase of cellular cAMP levels (by forskolin or IBMX) and increase of
`[Ca2⫹]i (with Bay K8664, A23187, or acetylcholine), and subsequent chromogranin A secretion. Basal release of chromogranin A amounted to
`11.8 ⫾ 4.3 ng/mL during a 25-minute incubation period and was significantly stimulated by each of these agents. The basal release (control) or
`chromogranin A was set at 100%.
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`Figure 7. Effects of guanylin, STa, and 8-Br-cGMP on the (A) regulated secretion and (B) uptake of GABA by BON cells. (A) BON cells released
`GABA in a regulated fashion when treated with either 50 mmol/L K⫹ or 1 ␮mol/L BayK 8664. Increases in cellular cGMP levels by guanylin, STa, or
`8-Br-cGMP also induced a significant increase of GABA secretion. In addition, dibutyryl-cAMP induced a significant increase in GABA release from
`BON cells. Basal GABA release (control) under nondepolarizing conditions was set at 100%. (B) GABA uptake in untreated BON cells (control) was set at
`100%. In the presence of 2 mmol/L unlabeled GABA, the uptake of labeled GABA was inhibited by about 87%, indicating that BON cells possess a
`specific GABA transport system. Neither 1 ␮mol/L guanylin and 100 ␮mol/L 8-Br-cGMP nor 1 ␮mol/L BayK 8644 inhibited GABA uptake.
`
`Discussion
`Guanylin is a well-established peptide regulator of
`epithelial chloride and fluid transport in intestinal and
`possibly also in pancreatic, neuroendocrine cells. Gua-
`nylin occurs in epithelial cells and seems to be secreted
`apically to act on its receptor, GC-C, located also in the
`apical plasma membrane of epithelial cells. Thereby,
`guanylin seems to function in an endocrine, paracrine,
`and even autocrine manner.5
`Using the pancreatic neuroendocrine cell line BON,
`we show for the first time that guanylin mediates
`regulated secretion of secretory granules (chromogranin
`A) and small synaptic vesicle analogues (GABA) via an
`elevation of cGMP. Our data are supported by the
`detection of functional guanylin receptors (GC-C) in
`BON cells. GC-C expression was shown by means of
`RT-PCR at mRNA and at protein levels by stimulated
`intracellular cGMP formation after either guanylin or STa
`treatment. The observed effects were mimicked by the
`membrane-permeable cGMP analogue, 8-Br-cGMP,
`strongly suggesting that the second messenger, cGMP,
`indeed is able to influence, besides fluid and chloride
`transport, vesicular release of peptides and neurotransmit-
`ters in neuroendocrine cells. In BON cells, the E. coli STa,
`another ligand of the guanylin receptor GC-C, was more
`potent than guanylin in cyclic GMP formation in
`accordance with previous studies on the intestinal epithe-
`lial cell line T84 and native intestinal mucosal cells.6,18,19
`Further characterization of the cGMP pathway in
`BON cells showed coexpression of GC-C together with
`
`GC-A, the latter being a receptor for natriuretic peptides,
`whereas GC-B and soluble GC were not detectable
`(Figure 2). The observed, considerable lower effects of the
`natriuretic peptides for the induction of cGMP formation
`compared with guanylin and STa (Figure 2) are best
`explained by a lower expression level of GC-A compared
`with GC-C in BON cells.
`It is noteworthy that the extent of the cGMP-activated
`secretion parallels changes in [Ca2⫹]i, a pathway crucial
`in regulated secretion of BON cells.10,11 The drastic
`activation of BON cell secretion by the adenylate cyclase/
`cAMP pathway, i.e., by forskolin and IBMX, is most
`likely explained by activation of a specific cAMP-
`dependent protein kinase and phosphorylation of target
`proteins involved in exocytosis. Furthermore, the ob-
`served high cGMP levels induced by guanylin and STa in
`pancreatic neuroendocrine cells may also activate cAMP-
`or cGMP-dependent protein kinases.20,21
`increases in
`In pancreatic acinar cells of the rat,
`intracellular cGMP have been reported to mediate di-
`rectly cellular Ca2⫹ uptake combined with a replenish-
`ment of agonist-sensitive Ca2⫹ stores.22,23 By contrast, in
`BON cells, guanylin and STa did not affect [Ca2⫹]i (this
`study). This suggests that pancreatic acinar cells express a
`signal transduction pathway linking cGMP levels to
`vesicular release, which is different from neuroendocrine,
`pancreatic cells. These cell type–specific differences within
`the pancreatic organ may also reflect the observed
`variable secretory responses to circulating or locally
`increased guanylin levels.5
`
`Roxane Labs., Inc.
`Exhibit 1040
`Page 006
`
`

`
`April 1998
`
`GUANYLIN–STIMULATED NEUROENDOCRINE SECRETION 797
`
`Due to its expression in neuroendocrine cells,5 gua-
`nylin may be released from secretory granules via the
`regulated secretory pathway of these cells. In addition,
`because guanylin was localized in enterocytes,5,24 the
`peptide seems to be secreted from these cells via an
`unidentified secretory pathway. It is therefore conceivable
`that release of guanylin from enterocytes and/or neuroen-
`docrine cells in turn affects secretion of various other
`peptide hormones and neurotransmitters.
`In disease, e.g., in neuroendocrine gastroenteropancre-
`atic tumors associated with hypersecretion syndromes
`unresponsive to somatostatin therapy, the observed Ca2⫹-
`insensitive vesicular release may be of pathophysiological
`importance. Due to excessive release of guanylin as well as
`possible, other unidentified factors, pharmacological con-
`trol of hypersecretion (e.g., with somatostatin analogues)
`remains ineffective because of the lack of pharmacological
`agents controlling Ca2⫹-independent regulated, secretory
`pathways.
`In summary, cGMP stimulates the release of both
`neuropeptides and neurotransmitters from neuroendo-
`crine secretory granules and small synaptic vesicle ana-
`logues, respectively. Ongoing studies focus on a detailed
`molecular dissection of the signal transduction cascade
`mediated by guanylin leading to vesicular release and
`cross talks between secretory pathways. This should
`result in alternative therapeutics from current treatment
`modalities.
`References
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`
`Received December 3, 1996. Accepted January 2, 1998.
`Address requests for reprints to: Bertram Wiedenmann, M.D.,
`Universita¨tsklinikum Charite´ der Humboldt-Universita¨t zu Berlin,
`Medizinische Klinik m. S. Hepatologie und Gastroenterologie, Cam-
`pus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin,
`Germany. Fax: (49) 30-45053902.
`Supported by Deutsche Forschungsgemeinschaft, the Deutsche
`Krebshilfe/Mildred Scheel Stiftung, and the Verum Stiftung.
`The authors thank Dr. C. M. Townsend, Jr., Department of
`Surgery, Medical Branch, University of Texas (Galveston, Texas), for
`his generous gift of BON cells;
`I. Eichhorn, E. Kock, and S.
`Haxelmans for their excellent technical assistance; and Drs. F.
`Hofmann and P. Ruth, Institut fu¨r Pharmakologie und Toxikologie,
`TU Mu¨nchen, for helpful d