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J. Biol. Chem., Vol. 280, Issue 20, 19576-19586, May 20, 2005
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From the
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo 05508-900, Brazil, the Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo 04023-062, Brazil, the ¶Departamento de Nefrologia, Universidade Federal de São Paulo, São Paulo 04044-020, Brazil, and ||Pediatric Cardiology, Cardiac Center Leipzig, University of Leipzig, 04280 Leipzig, Germany
Received for publication, March 7, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Most of the biological actions of BK and kallidin are mediated by a serpentine receptor coupled to a G-protein, the kinin-B2 receptor (B2BKR), which is constitutively expressed and widely distributed throughout central and peripheral tissues under physiological conditions. However, there is evidence that expression of B2BKRs during development is regulated. For instance, B2BKR expression has been shown to be involved in the development of the urinary and cardiovascular systems (10). Inhibition of B2BKR activity in rat embryos resulted in animals with disturbed kidney development (11). Besides being regulated during the ontogenesis of cardiovascular and urinary systems, a large set of evidence exists showing that modulation of B2 receptor expression and function also appears during neuronal development. Thus, it has been detected in central and peripheral noradrenergic neurons, in the spinal cord, in neuronal differentiating PC12 pheochromocytoma cells, and in neuroblastoma and glia-derived cell lines (12–18). A cross-talk between the B2BKR and other hormone and neurotransmitter receptors involved in neuronal development and function has been reported, such as endothelin, serotonin, and purinergic receptors (19, 20).
P19 is an embryonic carcinoma cell line that has been extensively validated as a model of in vitro neuronal differentiation by retinoic acid (RA) induction (21, 22). In these cells, other neuropeptide receptors such as the head activator receptor, the endothelin B receptor, and the substance P receptor are expressed during neuronal differentiation (23–25). The signal transduction pathways triggered by these receptors include transient increases in cytosolic-free calcium concentration [Ca2+]i by the mobilization of intracellular calcium stores and also induction of calcium influx from the extracellular environment. BK, acting via B2BKRs, also induces a transient increase in [Ca2+]i.
The modulation of B2BKR expression during the neuronal differentiation of cultured P19 cells was investigated herein at the levels of mRNA transcription, protein expression, and activity. Moreover, we have examined high and low molecular weight kininogen gene expression as precursors of the B2BKR agonists BK and kallidin as well as secretion of BK by differentiating P19 cells into the culture medium. The influence of the B2BKR antagonist HOE-140 has been tested on early and late stages of neuronal differentiation of P19 cells.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cell Culture and Neuronal Differentiation—P19 embryonal carcinoma cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Cultilab, Campinas, Brazil), 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate, in a humidified incubator at 5% CO2 and 37 °C. To induce neuronal differentiation of P19 embryonic carcinoma cells, cell cultures were stimulated to form embryonic bodies (EBs) in bacterial grade Petri dishes by addition of 1 µM "all-trans"-RA at a density of 5 x 105 cells/ml in defined medium (26). In order to avoid adhesion of the cell culture to plastic surfaces, dishes were coated with 0.2% agarose prior to cell plating. After 2 days, the EBs were plated in adherent culture flasks in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. After 48 h, the medium was replaced with defined medium to allow neuronal maturation. In order to prevent the increase of the percentage of glial cells in cell cultures older than 8 days, cultures were treated on day 8 with 50 µg/ml cytosine arabinoside to allow survival of only post-mitotic neurons.
Reverse Transcription and PCR—Total RNA was isolated using TRIzol (Invitrogen) from undifferentiated P19, EBs, and differentiated P19 neurons in different time periods. Integrity of the isolated RNA was assessed by separation on a 1% ethidium bromide-stained agarose gel. Contaminating DNA was removed using DNase I (Ambion Inc., Austin, TX) at a concentration of 1 unit/µg RNA in the presence of 20 mM Tris-HCl, pH 8.4, containing 2 mM MgCl2 and 50 mM KCl for 15 min at 25 °C.
Five µg of total RNA were used for cDNA synthesis in a total volume of 20 µl in the presence of 50 mM Tris-HCl, pH 8.3, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM dNTPs, and 50 ng of random primers with 200 units of RevertAidTM H Minus Moloney murine leukemia virus-reverse transcriptase (Fermentas Inc, Hanover, MD) for 50 min at 42 °C. For PCR amplifications, 5 µl of the reverse transcription reaction were used as a template. PCRs were performed in the presence of 2.5 mM MgCl2, 0.2 mM dNTPs, 20 µM each reverse and forward primers, and 0.5 units of Taq polymerase (Invitrogen). Primer sequences for amplification of B2BKR, 
-actin, M1–M3 muscarinic acetylcholine receptor (mAChR), and high and low molecular weight kininogen cDNAs are listed in Table I.
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For M1–M3 mAChR and -actin cDNA amplification, cycling conditions for PCR were 10 min at 95 °C, 35 cycles of 30 s at 94 °C, 20 s at 62 °C, 30 s at 73 °C, followed by a final step for 7 min at 73 °C.
PCR conditions for amplification of kininogen cDNAs were one initial cycle for 3 min at 94 °C, 36 cycles of 1 min at 94 °C, 20 s at 55 °C, 45 s at 72 °C, and a final elongation cycle for 5 min at 72 °C (27). PCR products were sequenced and confirmed as high and low molecular weight kininogen cDNAs. The PCR products were electrophoresed on a 2% agarose gel, visualized by ethidium bromide staining under UV illumination, and verified for their expected size (see Table I). The gel was scanned, and the band intensity was measured by densitometry (TINA software, Covance, North Yorkshire, UK). Variations in cDNA concentrations were normalized with -actin as an internal control, which is a constitutively expressed gene. Relative mRNA transcription was quantified as the x-fold increase of obtained densitometry values over threshold background levels.
Real Time PCR—Real time PCR was performed in order to quantify B2BKR mRNA transcription in embryonal P19 cells and P19 cells undergoing neuronal differentiation. Total RNAs (750 ng) were reverse-transcribed to cDNA as detailed above. The reaction product was amplified by real time PCR on the 7000 Sequence Detection System (ABI Prism, Applied Biosystems, Foster City, CA) using the Taqman PCR core reaction kit (PerkinElmer Life Sciences). The thermal cycling conditions composed an initial denaturation step of 95 °C for 10 min and 50 cycles at 95 °C for 15 s and 60 °C for 1 min. Experiments were performed in triplicates for each data point. B2BKR mRNA abundance was quantified as a relative value compared with an internal reference, -actin, whose abundance was believed not to change between the varying experimental conditions (28). Primers used for real time PCR are as follows: murine B2BKR (GenBankTM accession number NM009747), forward primer 5'-CCCTTCCTCTGGGTCCTCTT-3' and reverse primer 5'-GAAGAACACGCTGAGGACAAAGA-3'; 6-FAM probe, 5'-CCGCACTGGAGAAC-3'; murine -actin (GenBankTM accession number NM007393) forward primer 5'-CTGGCCTCACTGTCCACCTT-3' and reverse primer CGGACTCATCGTACTCCTGCTT-3'; 6-FAM probe, 5'-CTGATCCACATCTGCT-3'.
Quantitative values for B2BKR and -actin mRNA transcription were obtained from the threshold cycle number where the increase in the signal associated with an exponential growth of PCR products begins to be detected. Melting curves were generated at the end of every run to ensure product uniformity. The data were analyzed according to the manufacturer's instructions. B2BKR mRNA of undifferentiated and differentiated P19 cells was quantified as follows. The relative target gene expression level was normalized on the basis of -actin expression as an endogenous RNA control. 
Ct values of the samples were determined by subtracting the average Ct value of B2BKR mRNA from the average Ct value of the internal control -actin.
Immunochemical Staining and Confocal Laser-scanning Microscopy—For immunofluorescence detection undifferentiated P19 cells or EBs recovered after 48 h of induction to differentiation with retinoic acid were seeded (2.5 x 105 cells/ml) in chamber slides containing eight wells (Lab-Tek®, Naperville, IL). The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at 25 °C. For blocking of unspecific binding of the antibody, cells were incubated for 2 h with nonfat-milk (2%) containing 0.01% saponin and 2% rabbit serum. Then, the cells were incubated either with anti-NF200 rabbit (1:150) (Sigma), anti--III-tubulin rabbit (1:100) (Covance Research Products, Denver, PA), or anti-B2BKR goat antibodies (1:50) (Santa Cruz Biotechnology, Heidelberg, Germany). The slides were washed three times for 2 min each with 500 µl of nonfat-milk (2%) containing 0.01% saponin and 2% rabbit serum, and were then incubated with anti-goat IgG-Alexa-Fluor 488 secondary antibodies (Molecular Probes, Eugene, OR) to detect NF-200 expression, with anti-goat IgG-Cy5 (Zymed Laboratories Inc.) or anti-goat IgG-fluorescein isothiocyanate secondary antibodies (Abcam, Cambridge, MA) for detection of B2BKR, or with anti-rabbit IgG-Cy3 (Abcam) for detection of -III-tubulin for 30 min at room temperature (at the dilutions recommended by the manufacturer) in PBS containing 2% bovine serum albumin. Counterstaining of cell nuclei was done with 10 µg/ml 4',6-diamidino-2-phenylindole (DAPI). Following washing with PBS, coverslips were mounted on the slides with Vectashield (Vector Laboratories, Burlingame, CA), and slides were examined using the Leica Owin program (Leica Microsystems, Bannockburn, IL) and a ADIMEC MX12P camera coupled to a Optiphot-2 epifluorescence microscope (Nikon, Melville, NY).
Confocal laser-scanning microscopy was used to determine whether the B2BKR and NF-200 are expressed by the same cells. Double-immunofluorescence staining against the B2BKR and NF-200 was done as detailed above. Cells were observed under an inverted laser-scanning microscope (Zeiss LSM 510, Zeiss, Jena, Germany) using a Zeiss Plan-Apo 63x 1.4 NA oil immersion objective lens. The Alexa-Fluor 488 (Molecular Probes) was excited by using a 488-nm argon laser and NF-200 immunofluorescence emission was detected with a 515–540-nm bandpass filter. Cy5 (Zymed Laboratories Inc.) fluorescence was excited at 647 nm using the helium-neon laser, separated from the argon laser line by a FT 510 beam splitter. B2BKR immunofluorescence emission above 655 nm was collected using a long pass filter (RG 665). Control experiments were conducted to exclude any unspecific fluorescence emission. Instead of double-excitation at 488 nm and 647 nm, the absence of fluorescence emission above 655 nm following excitation at 488 nm was verified. No fluorescence emission above 665 nm was detected when only NF-200 immunofluorescence had been excited (data not shown).
Western Blot Analysis—Partially purified membranes of P19 embryonic cells and EBs following induction to neuronal differentiation were prepared by cell sonication and differential centrifugation. One hundred million cells were collected from culture dishes, washed three times in PBS containing 100 µM phenylmethylsulfonyl fluoride and 0.5 µg/ml leupeptin, and sonicated on ice until at least 95% of the cells were lysed. The cell extract was centrifuged at 600 x g for 10 min and 4 °C to remove cell debris. The supernatant was centrifuged at 100,000 x g for 1 h. The obtained plasma membranes were washed twice with PBS containing protease inhibitors and then resuspended in SDS-loading buffer at a concentration of 1 mg/ml protein. For Western blotting, membrane proteins (40 µg) and molecular weight markers were denatured by boiling for 5 min and then separated by 12% SDS-PAGE according to the Laemmli method (29). The proteins separated on the gel were electrophoretically transferred at 400 mA for 80 min onto a nitrocellulose membrane (Transblot, 0.45 µm, Bio-Rad). The nitrocellulose membrane was stained with 0.1% Ponceau solution in order to verify the transfer of the proteins and to localize the migration of the molecular weight markers. The nitrocellulose membrane was destained and blocked for 2 h with Tris-buffered saline with 0.03% Tween (TBS-T) containing 3% nonfat milk prior to the addition of a 1:200 dilution of a goat polyclonal anti-B2BKR antibody (Santa Cruz Biotechnology). Following 14 h of incubation with the primary antibody at 4 °C, the membrane was washed three times for 15 min with TBS-T. The membrane was blocked again for 30 min with TBS-T containing 3% nonfat milk and then incubated for 2 h at room temperature with a 1:1,000 dilution of a secondary anti-goat IgG antibody. The labeled proteins were detected by using the Super Signal West Pico Chemiluminescent Substrate (Pierce) according to the instructions provided by the manufacturer.
Quantification of BK in Culture Medium from Differentiating P19 Cells by High Performance Liquid Chromatography (HPLC)—Cell culture supernatants from P19 cells from days 0 to 7 following induction to differentiation were used for quantification of BK concentration as described elsewhere (30).
Protease inhibitors were added immediately following collection of culture supernatants to final concentrations of 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM p-hydroxymercuribenzoate, 1.5 mM o-phenanthroline, and 1 µM pepstatin. Cell culture supernatants were frozen in liquid nitrogen and stored at -80 °C until further use. For BK extraction, 2 ml of cell culture supernatant were loaded on a Sep-Pak C18 cartridge (Waters, Milford, MA), which had been equilibrated in 5% acetonitrile and 0.1% H3PO4. The cartridges were eluted with 3 ml of 35% acetonitrile in 0.1% H3PO4. The pre-purified extracts were dried in a Speedvac concentrator (Boc Edwards, Wil-mington, MA), resuspended in 500 µl of 0.1% H3PO4 containing 5% acetonitrile (phase A), and then used for HPLC. For chromatographic identification and quantification of BK, 100 µl of the extracted sample was injected into an HPLC system (Milton Roy, Ivyland, PA), and the separation was performed through a reverse-phase RP18 Brownlee column (4.6 x 250 mm, Millipore, Billerica, MA) equilibrated with 0.1% H3PO4 containing 5% acetonitrile (phase A). After 5 min of isocratic elution, peptides were eluted with a 0–35% linear gradient (v/v) of 90% acetonitrile in 0.1% H3PO4 (phase B) developed for 20 min at a flow rate of 1.5 ml/min. The effluent was monitored by absorbance at 214 nm. BK was identified by elution position and quantified by peak area integration using synthetic BK as a standard.
Calcium Measurements in Single Cells by Confocal Microscopy— Measurements of [Ca2+]i in undifferentiated and neuronal differentiated P19 (embryonic carcinoma) cells on days 7 and 8 following induction with RA were performed by using LSM 510 confocal microscope (Zeiss, Jena, Germany). Two hundred fifty thousand cells were seeded and allowed to attach on cover slides in p60-mm culture dishes (Nalge Nunc International, Rochester, NY) 48 h before the measurements. Cells were loaded with 4 µM fluo-3AM (Sigma) in 0.5% Me2SO and 0.1% of the nonionic surfactant pluronic acid F-127 for 30 min at 37 °C, washed three times with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, and transferred to defined medium. Fluo-3AM fluorescence was excited at 488 nm, and fluorescence was measured at 515 nm using a bandpass filter. [Ca2+]i of undifferentiated cells was measured for 10 s. Then 1 µM BK or 1.5 mM carbamoylcholine (CCh) was added, and changes in [Ca2+]i were monitored for a further 2 min. Images were collected every 1.5 s. At the end of each experiment, 5 µM ionophore (4-Br-A23187) followed by 10 mM EGTA were used to determine maximal (Fmax) and minimal (Fmin) fluorescence values, respectively. The experimental intracellular calcium concentrations from relative fluorescence values (F) obtained upon BK addition were calculated using the following formula: [Ca2+]i = Kd(F - Fmin)/(Fmax - F), assuming a 450 nM Kd for fluo-3AM (31). Concentrations were calculated for cell populations containing at least each 10 cells.
Treatment of P19 Cells with HOE-140 at the Initial Stage of Differentiation—P19 cells at a density of 105 cells/ml were seeded in nonadherent cell culture dishes and treated with RA (1 µM) and increasing concentrations (0–1 µM) of the B2BKR antagonist HOE-140 (32) at intervals of 24 h. After 48 h, 30 randomly chosen areas of each cell culture dish were photographed, and the sizes of EBs were measured and compared with EBs of cell cultures that had been kept in the absence of HOE-140.
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For evaluation of HOE-140 (1 µM) pretreatment on day 5 on cholinergic signal transmission at days 7 and 8, HOE-140 was removed from the cell culture 6 h prior to calcium measurements, by medium change and washing the cell layers twice. Control cultures in absence of HOE-140 were treated the same way. Lys-des-Arg9-Leu8-BK, a kinin-B1 receptor antagonist, which does not bind to the B2BKR (33), was used as control at 1 µM concentration to confirm the specificity of HOE-140 action.
Determination of the Participation of Muscarinic Acetylcholine Receptor in Carbamoylcholine-induced Changes in [Ca2+]i—For studying muscarinic receptor-mediated intracellular Ca2+ mobilization upon carbamoylcholine stimulation, P19 cells on day 8 of neuronal differentiation were pretreated for 30 min with 180 ng/ml thapsigargin prior to calcium measurements in order to deplete the intracellular calcium stores and inhibit muscarinic receptor-induced calcium mobilization.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-III-tubulin (Fig. 1F). In order to avoid contamination of elder P19 neuron cell cultures with glial cells that start to appear on day 8 after induction to neuronal stimulation (23), cells were treated on day 8 with the DNA synthesis inhibitor cytosine arabinoside (50 µg/ml) that eliminates glial cells in proliferation but not differentiated post-mitotic neurons.
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Expression of High and Low Molecular Weight Kininogens and Secretion of Bradykinin by P19 Cells—High and low molecular weight kininogens were generated by alternative splicing from a single kininogen gene (27). The presence of mRNA transcription of high and low molecular weight kininogens during neuronal differentiation of P19, which give origin to bradykinin and kallidin, respectively, was detected by RT-PCR. High molecular weight kininogen 1 transcription could be detected throughout the course of neuronal differentiation (Fig. 4A). Three sizes of kininogen 1 mRNA, corresponding to sizes of 1000, 752, and 400 nucleotides, derived from alternative splicing were detected. Kininogen 2 mRNA, also derived from alternative splicing, was present in low quantities, beginning from day 2 of differentiation. Low molecular weight kininogen mRNA transcription, giving origin to the B2BKR agonist kallidin, was present in low quantities beginning from day 2.
Secretion of BK into the P19 cell cultures in neuronal differentiation was quantified by HPLC analysis. Undifferentiated P19 cells did not liberate any BK into the culture medium, whereas at the beginning from the stage of EB formation and during later neuronal differentiation the amount of BK detected in the culture medium increased (Fig. 4B). Maximal values of 0.7 ± 0.1 nmol BK/106 cells were detected on day 7 when cells undergo final neuronal maturation.
The Expression of Functional B2BKRs—The functionality of B2BKRs expressed in P19 cells was determined by measuring changes in [Ca2+]i following stimulation of the cells with bradykinin (1 µM) by confocal microscopy using the calcium-sensitive fluorophore fluo-3AM (Fig. 5). The undifferentiated embryonal P19 cells did not respond to BK application with a change in [Ca2+]i (Fig. 5, A and D). Following induction to neuronal differentiation and aggregation, the cells responded to BK application with an elevation of [Ca2+]i. Maximal responses were found in cells after 7 and 8 days of neuronal differentiation (Fig. 5, A and D). Fig. 5D shows a representative Ca2+ response of single undifferentiated and differentiated cells. We found that responsive cells showed an average increase in [Ca2+]i of 900 ± 200 nM (Fig. 5E).
Inhibition of Carbamoylcholine-induced Calcium Mobilization in Differentiating Neurons That Had Been Pretreated with HOE-140 —P19 neuronal precursors stimulated to in vitro differentiation became post-mitotic after 4 or 5 days following RA addition (34) and underwent neuronal maturation and end-terminal differentiation, such as the formation of synapses and the expression of functional neurotransmitter and neuropeptide receptors. mAChR expression has been demonstrated in differentiated P19 neurons (35) and has been implicated in the regulation of final neuronal differentiation such as neurite outgrowth (36). Fig. 5B shows that the calcium response to CCh, which is already present in embryonal P19 cells, is largely increased following 7 and 8 days of neuronal differentiation (Fig. 5, B and D, lower panels).
In order to evaluate the effect of the B2BKR antagonist HOE-140 on final neuronal maturation, at the example of muscarinic receptor activity, P19 cells in neuronal differentiation were pretreated on day 5 with HOE-140 (1 µM). Control experiments were carried out in which P19 cells were differentiated in the presence of the kinin-B1 receptor antagonist Lys-des-Arg9-Leu8-BK (33). Following removal of the B2BKR antagonist HOE-140 or the control peptide Lys-des-Arg9-Leu8-BK 6 h prior to the calcium measurements, P19 cells were tested for B2BKR and muscarinic receptor activity on days 7 and 8 of differentiation (Fig. 5). P19 cells differentiated in the presence of HOE-140 responded to BK application (1 µM) with a smaller increase in [Ca2+]i than did the untreated control cells, suggesting a down-regulation of B2BKRs because of the prolonged presence of the antagonist (Fig. 5A). When in control experiments, differentiated P19 cells were treated with HOE-140 and then washed for 75 min with cell culture medium, the BK-induced calcium response was fully restored (Fig. 5C).
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Fig. 5E gives a quantification of Ca2+ responses upon stimulation of HOE-140-pretreated and control P19 cells on day 7 by BK or CCh. The calcium responses upon stimulation of Lys-des-Arg9-Leu8-BK-pretreated P19 cells with CCh compared with responses of untreated control cells were largely unchanged (Fig. 5E), demonstrating that HOE-140-induced inhibition of muscarinic receptor function is because of the B2BKR blockade and not to possible toxic effects of a high concentration of the antagonist.
Down-regulation of mAChR mRNA Transcription Following Pretreatment of Differentiating P19 Cells with HOE-140 —On day 5 following stimulation to neuronal differentiation by RA, B2BKR activity was blocked by addition of HOE-140 (1 µM). HOE-140 pretreatment resulted in a decrease of B2BKR activity, as shown by calcium measurements in the presence of BK. Pretreatment with HOE-140 also resulted in a decrease of M1–M3 muscarinic receptor mRNA transcription on day 8 of differentiation as determined by semi-quantitative RT-PCR (Fig. 6A). The analysis of intensities of the bands of the ethidium bromide-stained RT-PCR products of RNA isolated from P19 cells, which had been pretreated with HOE-140, revealed a decrease of M1 and M2 mAChR and M3 mAChR transcription levels by 96 and 93% compared with M1–M3 mAChR mRNA transcription levels of untreated control cells, respectively (Fig. 6B). The decrease in mAChR activity following HOE-140 pretreatment correlated with a down-regulation of gene expression of M1–M3 receptor subtypes. In the absence of HOE-140, gene expression levels of muscarinic receptors increase until day 8 of differentiation, as shown by the example of the M3 mAChR (Fig. 6C).
Inhibition of Formation of EBs in the Presence of the B2BKR Antagonist HOE-140 —In order to evaluate the implication of the B2BKBR during early neuronal determination and differentiation, embryonic P19 undifferentiated cells were plated in Petri dishes in the absence and in the presence of 10 nM to 1 µM HOE-140 and stimulated with 1 µM RA for EB formation. The sizes of EBs were compared after 2 days of treatment with the control experiment, where only RA was used for stimulation of neuronal differentiation (Fig. 7). Fig. 7A shows the results of a representative experiment. The percentage of EBs with sizes less than 1 mm increased in the presence of HOE-140, whereas large EBs mostly disappeared. Fig. 7B demonstrates that in presence of HOE-140 the overall diameters of EBs are reduced by an average of 20% as compared with the uninhibited controls. The differences in sizes of treated and untreated EBs were statistically significant at HOE-140 concentrations of 10 nM to 1 µM (p < 0.05) (Fig. 7C). The treatment of undifferentiated, proliferating cells for 48 h with 1 µM HOE-140 did not result in a reduction of cell number, indicating that HOE-140 did not have an unspecific toxic effect on the cell population (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B2BKRs have been shown to be expressed in neuronal determined cell cultures and also in the brain (42, 43). The existence of a mechanism of regulation of B2BKR gene expression by trans-acting factors to a silencer motif in the B2BKR promoter has been shown in the neuronal cell line NG108-15 (44).
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The cell line P19 (21) was established previously as a model for in vitro neuronal differentiation. P19 cells constitute those cells of the inner cell mass of the blastocyst during the initial period of development, and the differentiation of P19 cells is believed to closely mimic critical events during early embryogenesis. RA treatment induces P19 cells to develop a neuronal phenotype characterized by the expression of neurotransmitters, the formation of neurites with synaptic specializations, and the expression of the neuronal markers -III-tubulin and low (NFL), medium (NFM), and high (NFH) molecular weight neurofilament subunits (48–50). The NFL and NFM subunits are expressed during the early stages of neuronal differentiation, whereas NFHs such as neurofilament 200 (NF-200) appear later on days 5 and 6 when neurons are already post-mitotic, reaching maximal expression following neuronal maturation on day 8. We have detected B2BKR expression and mRNA transcription during the course of neuronal differentiation. The highest levels of receptor expression and mRNA transcription were detected on days 8 and 9 of differentiation. P19 neurons responded to BK application with a large increase in [Ca2+]i, whereas undifferentiated cells did not respond to BK stimulation (Fig. 5). The absence of a visible response of undifferentiated P19 cells to BK application may be explained by a low concentration of B2BKR expression on the cell surface. The absence of a response to BK application may also result from the existence of post-translational modifications that regulate B2BKR configuration and activity (51).
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It was found that inhibition of B2BKR action at the early stage of differentiation during the formation of EBs counteracts differentiation as determined by the reduced size of EBs (20% versus control) (Fig. 7). Supposedly, B2BKR action is needed to induce calcium transients during early development, known to be necessary for following differentiation processes.
The effect of B2BKR blockade has also been examined on later differentiation events, such as the expression and activity of the mAChR (Figs. 5 and 6) whose functions are implicated in neurite outgrowth and formation of functional synapses. P19 cells have been proposed to serve as an in vitro neuronal differentiation model for studying mAChR subtypes in a proper neuronal context, as differentiated P19 neurons express high concentrations of cholinergic markers as well as subtypes of the muscarinic receptor family (35, 53).
Cholinergic function has been suggested to play a key role in cell-cell communication during migration and differentiation of neural precursor cells. Acetylcholine is released by the growth cone in the process of fiber elongation, and fiber outgrowth in several systems is modulated by acetylcholine (55). Stimulation and inhibition of mAChR activity led to a higher and lower ability of differentiating neuroblastoma cells to induce neurite outgrowth and synapsin I expression, indicating its importance in neuronal maturation (35). Muscarinic and other hormone and neurotransmitter receptor expression is regulated during in vitro neuronal differentiation. For instance, RA induction of neuroblastoma cells to neuronal differentiation results in an increase of muscarinic receptor expression (56). We have shown that the M3 receptor gene expression increases during the course of neuronal differentiation (Fig. 6C). P19 cells on day 8 of differentiation responded with a higher increase in [Ca2+]i to CCh stimulation than cells on day 5 did (data not shown). Similar results were obtained from cells on days 5 and 8 of differentiation that were stimulated with N-methyl-D-aspartic acid or endothelin-B (data not shown). In accordance with these data, P19 cell cultures in neuronal differentiation showed increased responsiveness to application of 
-aminobutyric acid when they were maintained in differentiation for more than 5 days (57). Synaptotagmin, indicating the development of functional synapses, was detected on day 9 of differentiation of P19 and was absent on day 6 (53).
It was herein found that pretreatment of P19 cells in neuronal differentiation with the B2BKR antagonist HOE-140 at 1 µM concentration resulted in less gene expression of M1–M3 mAChR on day 8 and a partial loss of mAChR function on days 7 and 8 of neuronal differentiation. These observations indicate that P19 cells at the time point of HOE-140 addition on day 5 of differentiation, although already expressing neuronal markers such as high molecular weight neurofilaments, are yet in the stage of neuronal precursors undergoing final maturation, which is inhibited in the presence of HOE-140.
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Previously published work supports the hypothesis of B2BKR participation in neuronal differentiation. In PC12 cells, BK signaling resulted in translocation of protein kinase C
, an enzyme implicated in regulation of neurite outgrowth, and therefore BK was suggested to participate in neuronal differentiation of this cell line (59). The BK-induced signal transduction closely resembles inositol 1,4,5-trisphosphate accumulation followed by calcium release from intracellular stores, which was characterized during differentiation of the human neuroblastoma cell line SH-SY5Y (60, 61). Calcium transients have also been observed in the growth cones of developing neurons (62). It seems likely that B2BKR function is needed during neuronal maturation of P19 cells in vitro. This hypothesis is supported by published work (63, 64) showing BK-induced neurotransmitter release in differentiating PC12 and neuroblastoma cells. Bush et al. (64) observed that short and long term treatment of PC12 cells with nerve growth factor increased the potency of BK to raise intracellular calcium concentration [Ca2+]i 10- and 100-fold, respectively, implicating a crucial function of BK in inducing fast calcium waves in differentiating PC12 neurites (65).
We have shown that gene expression of kininogens, the precursors of the B2BKR agonists BK and kallidin, is induced following stimulation of P19 cells by RA and secretion of BK in the culture medium increases during the course of neuronal differentiation (Fig. 4). These results suggest the presence of an autocrine loop involving secretion of BK by P19 cells in order to activate B2BKRs on their cell surface and functional B2BKRs as prerequisites for the progress of neuronal differentiation. Both neuronal maturation and survival of P19 neurons are directly correlated with B2BKR activity. When differentiating P19 were treated over 3 days with the B2BKR antagonist HOE-140, less cells survived until day 8 (data not shown).
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In summary, the B2BKR is the first neuropeptide receptor whose function was directly related to neuronal differentiation by a functional assay. We have presented data showing that both BK secretion and B2BKR expression and activity are augmented during the course of neuronal differentiation. The fact that blockade of B2BKR function by the specific B2BKR antagonist HOE-140 inhibits the progress of early and later neuronal differentiation is taken as evidence that B2BKRs have a crucial role in neuronal differentiation rather than only being a marker of differentiation states. The presented study may encourage further investigations to understand the molecular mechanism implicated in the B2BKR activation and its role during neuronal differentiation, neurodegeneration, and neuroregeneration following brain injury.
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FOOTNOTES |
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** To whom correspondence should be addressed: Dept. de Bioquímica, Instituto de Química, Universidade de São Paulo, Caixa Postal 26077, CEP 05513-970, São Paulo, Brazil. Tel.: 55-11-3091-3810 (ext. 223); Fax: 55-11-3815-5579; E-mail: henning{at}iq.usp.br.
1 The abbreviations used are: BK, bradykinin; B2BKR, kinin-B2 receptor; CCh, carbamoylcholine; [Ca2+]i, cytosolic-free calcium concentration; DAPI, 4',6-diamidino-2-phenylindole; dd-H2O, double-distilled water; Me2SO, dimethyl sulfoxide; EB, embryonic body; NF, neurofilament; NFH, high molecular weight neurofilament; NFM, intermediate molecular weight neurofilament; kininogen L, low molecular weight kininogen; NFL, low molecular weight neurofilament; mAChR, muscarinic acetylcholine receptor; PBS, phosphate-buffered saline; RA, retinoic acid; HPLC, high performance liquid chromatography.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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