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Originally published In Press as doi:10.1074/jbc.M502513200 on March 14, 2005

J. Biol. Chem., Vol. 280, Issue 20, 19576-19586, May 20, 2005
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Neuronal Differentiation of P19 Embryonal Carcinoma Cells Modulates Kinin B2 Receptor Gene Expression and Function*

Antonio Henrique B. Martins{ddagger}, Rodrigo R. Resende§, Paromita Majumder§, Marcella Faria§, Dulce E. Casarini¶, Attila Tárnok||, Walter Colli§, João Bosco Pesquero{ddagger}, and Henning Ulrich, Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Project No. 2001/08827-4, Conselho Nacional de Desenvolvimento Científico, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil§**

From the §Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo 05508-900, Brazil, the {ddagger}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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Kinins are vasoactive oligopeptides generated upon proteolytic cleavage of low and high molecular weight kininogens by kallikreins. These peptides have a well established signaling role in inflammation and homeostasis. Nevertheless, emerging evidence suggests that bradykinin and other kinins are stored in the central nervous system and may act as neuromediators in the control of nociceptive response. Here we show that the kinin-B2 receptor (B2BKR) is differentially expressed during in vitro neuronal differentiation of P19 cells. Following induction by retinoic acid, cells form embryonic bodies and then undergo neuronal differentiation, which is complete after 8 and 9 days. Immunochemical staining revealed that B2BKR protein expression was below detection limits in nondifferentiated P19 cells but increased during the course of neuronal differentiation and peaked on days 8 and 9. Measurement of [Ca2+]i in the absence and presence of bradykinin showed that most undifferentiated cells are unresponsive to bradykinin application, but following differentiation, P19 cells express high molecular weight neurofilaments, secrete bradykinin into the culture medium, and respond to bradykinin application with a transient increase in [Ca2+]i. However, inhibition of B2BKR activity with HOE-140 during early differentiation led to a decrease in the size of embryonic bodies formed. Pretreatment of differentiating P19 cells with HOE-140 on day 5 resulted in a reduction of the calcium response induced by the cholinergic agonist carbamoylcholine and decreased expression levels of M1–M3 muscarinic acetylcholine receptors, indicating crucial functions of the B2BKR during neuronal differentiation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bradykinin (BK)1 and kallidin are biological active peptides generated by the proteolytic cleavage of kininogens by serine proteases of the kallikrein family. High molecular weight kininogens are precursors of BK, whereas low molecular weight kininogens give origin to kallidin. Along with other kinins, BK and kallidin elicit a wide range of physiological responses, being classically involved in the control of cardiovascular homeostasis and inflammation. As a matter of fact, altered function of the kallikrein-kinin system has been implicated in the development of various pathological conditions such as arthritis, pancreatitis, and asthma (for a review see Refs. 1 and 2). Emerging evidence shows that kinins are stored in neuronal cells of the central nervous system and may act as neuromediators in various functions, including the control of nociceptive information (for a review see Ref. 3). Expression of kallikrein in developing rat brains (4) supports the notion that kinin-induced receptor activity might be required during neuronal development. BK has also been shown to enhance the release of neurotransmitters such as noradrenalin and neuropeptide Y by sympathetic neurons, chromaffin cells, and pheochromocytoma cells (5–8). Moreover, BK implication in the control of calcium homeostasis has already been demonstrated in adult sensory neurons (9).

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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—If not otherwise indicated, all reagents were purchased from Sigma.

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, {beta}-actin, M1–M3 muscarinic acetylcholine receptor (mAChR), and high and low molecular weight kininogen cDNAs are listed in Table I.


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  		TABLE I
Primer sequences used for PCR amplifications

 
For B2BKR cDNA amplification, the reaction was cycled 35 times (95 °C for 45 s, 56 °C for 45 s, and 72 °C for 105 s) plus a final extension of 10 min at 72 °C. The 627-bp PCR product was gel-purified and DNA sequenced and confirmed to be identical to mouse B2BKR cDNA.

For M1–M3 mAChR and {beta}-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 {beta}-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, {beta}-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 {beta}-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 {beta}-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 {beta}-actin expression as an endogenous RNA control. {Delta}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 {beta}-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-{beta}-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 {beta}-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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  		FIG. 1.
Neuronal differentiation of P19 embryonal carcinoma cells. In vitro differentiation of P19 embryonal cells resembles processes during early development in vivo. Pluripotent P19 cells (A) are treated with RA and plated into nonadherent culture dishes to form EBs (B). Forty-eight h after induction to neuronal differentiation, EBs are collected and replated in adherent cell culture dishes. On day 5 (C), neuronal precursors become post-mitotic and undergo end-terminal differentiation that is completed on day 8 (D). Beginning on day 5, the developing neurons express NF-200, reaching maximal expression on day 8. E and F, immunostaining of neuron-specific proteins: differentiating P19 cells on day 8, expressing NF-200 and {beta}-III-tubulin, respectively. Cell nuclei were counterstained with DAPI. Images of immunostaining and DAPI staining were overlaid.

 
Treatment of P19 Cells in Later Differentiation (Days 5–8) with HOE-140 —For determining the effect of B2BKR inhibition on expression of mAChRs, 1 µM HOE-140 was added to the cells on day 5 following induction to differentiation and applied again at intervals of 24 h. Control reactions were set up in the absence of HOE-140. On day 8, total RNA was isolated from the cell cultures, and M1, M2, and M3 mAChR mRNA transcription was quantified by semi-quantitative RT-PCR as detailed above.

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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of B2BKRs and Neuron-specific Proteins during RA-induced Differentiation of P19 Cells—Murine P19 embryonal carcinoma cells were induced to the neuroectodermal differentiation by culturing free floating EBs in defined medium containing 1 µM RA (26) (Fig. 1, A and B). After 2 days, EBs were plated into tissue culture flasks, whereupon extensive morphological differentiation occurred after RA treatment (Fig. 1C). After 7 days, cells with neuron-like morphology appeared (Fig. 1D), reaching maximum expression levels of neuron-specific proteins after 7–9 days as detected by staining with antisera against NF-200 (Fig. 1E) and {beta}-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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  		FIG. 2.
Detection of B2BKR expression in P19 cells during neuronal differentiation. A, Western blot. Forty µg each of partially purified membranes of embryonal, undifferentiated P19 cells on day 0 and EBs on day 2 following treatment with RA were separated by SDS-PAGE. The proteins were transferred to a nitrocellulose membrane. The membrane was incubated with a polyclonal goat anti-B2BKR antiserum. The reaction was developed using the ECL reaction. Both undifferentiated cells and EBs express the B2BKR protein (45 kDa). B, detection of differential expression of B2BKRs by immunofluorescence. Following fixation, cells were incubated with an anti-polyclonal B2BKR antibody: undifferentiated P19 cells (UN, left panel), P19 cells at day 7 (middle panel), and day 9 (right panel) following induction to neuronal differentiation. A fluorescein isothiocyanate-conjugated secondary antibody was used to detect the expression of B2BKR. Counterstaining of cell nuclei was performed using DAPI. The expression of B2BKRs in undifferentiated cells was below the detection limit. Increasing percentages of cells expressing B2BKRs were detected on day 7 and 9 of neuronal differentiation. Scale bars indicate a size of 40 µm. C, detection of co-expression of B2BKR and NF-200. For double-immunofluorescence staining of P19 cells on day 8 of differentiation, incubation of fixed cells with primary antibodies against the B2BKR and NF-200 was performed as detailed above, using Cy5-labeled (Zymed Laboratories Inc.) and Alexa-Fluo 488 (Molecular Probes)-labeled secondary antibodies, respectively, for detection. Fluorescence emissions were separated by using a confocal microscope. The left and middle panels show the images collected for B2BKR and NF-200 expression in the same cells. In the right panel, images are overlaid, indicating that B2BKRs are expressed by neuronal differentiated cells.

 
P19 undifferentiated embryonal carcinoma cells and EBs express the B2BKR, as verified by Western blot analysis (Fig. 2A). Expression of the receptor is increased after cell aggregation following stimulation with RA and plating for morphological differentiation (Fig. 2B). The peak of maximal expression of B2BKR correlated with the expression of neuron-specific proteins as determined by immunofluorescence staining against NF-200. Double-immunostaining against the neuron-specific protein and B2BKR on day 8 of differentiation revealed that the B2BKR is expressed by neuronal differentiated cells (Fig. 2C).



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  		FIG. 3.
Detection and quantification of B2BKR expression during P19 neuronal differentiation by RT-PCR. A, real time PCR. For quantification of differential transcription of B2BKR mRNA, total RNAs from P19 in several stages of neuronal differentiation were prepared, and B2BKR mRNA was analyzed by reversed transcription followed by real time PCR (Taq-Man) as described under "Experimental Procedures." The relative expression levels were corrected with the internal control {beta}-actin. The graphics show B2BKR mRNA transcription levels in differentiating P19 cells compared with those in undifferentiated P19 cells. Cells on day 10 of differentiation were pretreated on day 8 with cytosine arabinoside, in order to avoid contamination of the cell cultures with increasing concentrations of proliferating glial cells. UN, embryonal P19 cells (n = 3); EB, embryonic bodies on day 2 of differentiation (n = 3); days 4 and 6 (n = 3, respectively); days 7 and 8 (n = 6, respectively); day 10 (n = 2). D, day of differentiation. The plotted data represent the mean values of n independent values ± S.D. B, RT-PCR, the upper panel shows the amplification of a cDNA corresponding to the B2BKR of total RNA collected from cells of day 8 of differentiation. Blank, RT-PCR was performed in the absence of RNA template. The amplified cDNA was confirmed as B2BKR cDNA by DNA sequencing.

 
RT-PCR and Real Time PCR—The B2BKR is continuously expressed during neuronal differentiation, but its mRNA transcription level varies during the time course of differentiation as determined by real time PCR (Fig. 3A). The highest levels of B2BKR mRNA were detected on day 8 of differentiation. Fig. 3B shows the PCR amplification of a fragment of B2BKR cDNA, obtained by reverse transcription, with an expected length of 627 nucleotides. The amplified cDNA was confirmed to be part of B2BKR cDNA by DNA sequencing. In undifferentiated cells and in cells 2 days after induction to neuronal differentiation, basal levels of B2BKR mRNA were detected by RT-PCR. Real time PCR revealed that there was no significant alteration in B2BKR mRNA transcription during early neuronal differentiation. Following replating of EBs in adherent culture plates on day 3, maximal levels of B2BKR mRNA transcription were reached after 8 days following addition of RA (Fig. 3A). On day 8 of differentiation, mRNA levels were 20-fold increased compared with those of undifferentiated cells. In already differentiated neurons on day 10, B2BKR mRNA levels decreased compared with those of day 8, implicating the crucial function of B2BKR expression during neuronal maturation that is complete before day 10.

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. 4.
Determination of kininogen mRNA transcription and quantification of bradykinin secretion in cell culture medium during neuronal differentiation in vitro. A, determination of high and low molecular weight kininogen mRNA transcription. Gene expression of kininogens was detected by RT-PCR using total RNA extracted from various days of differentiation. RT-PCR procedures are detailed under "Experimental Procedures." Products from alternative splicing from a single kininogen precursor mRNA corresponding to high molecular weight kininogens 1 and 2 and low molecular weight kininogens were amplified using primers detailed in Table I. Mouse brain (MB) and mouse liver (ML) were used as positive controls. UN, undifferentiated P19 cells; kininogen L, low molecular weight kininogen. B, quantification of BK in culture medium from differentiating P19 cells. Cell culture supernatants from differentiating P19 cultures (days 0–7 following addition of RA) were collected and analyzed for BK content by reversed phase HPLC. Synthetic BK was used as a standard for elution and quantification. The results represent mean values ± S.D. of three independent experiments.

 
The calcium response to the cholinergic agonist CCh was decreased by 90 and 50% on days 7 and 8, respectively, when cells had been differentiated in the presence of HOE-140 (1 µM) (Fig. 5, B and D). The CCh-induced rise in [Ca2+]i was confirmed to result from muscarinic, and not nicotinic, receptor activation, as the response was inhibited following pretreatment of the cells with thapsigargin (Fig. 5B) (37).

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).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
BK was originally characterized as a peptide with vasodilatory action and as a mediator during inflammatory processes. Besides its function in cardiovascular regulation, BK was also shown to elicit mitogenesis and the synthesis of DNA and is involved in the regulation of mitogenesis in arterial smooth muscle cells and in the proliferation of tumors (38, 39). In some tumors, such as in prostate cancer, BK acts as a growth factor (40), whereas in other tumor cells the inhibition of the B2BKR by the specific antagonist HOE-140 resulted in proliferation (41), leaving the physiological role of bradykinin during proliferation and differentiation processes undefined.

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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  		FIG. 5.
Imaging of changes in [Ca2+]i in P19 following stimulation by bradykinin and carbamoylcholine. Four µM fluo-3AM in 0.5% of Me2SO and 0.1% of pluronic acid were used to load the cells 30 min before addition of 1 µM BK or 1.5 mM CCh. Calcium imaging was performed by confocal microscopy. Following imaging of variations in [Ca2+]i of an unstimulated cell population, agonist was added, and stimulation was recorded for 3 min by collecting images in time intervals of 3 s. F, fluo-3AM fluorescence following addition of the agonist; F0 = Fluo-3AM fluorescence of the unstimulated cell population. Arrows mark the time point of ligand addition. A, changes in [Ca2+]i following stimulation by BK. Curve 1, P19 cells on day 8 of differentiation; curve 2, P19 cells on day 8 following pretreatment with HOE-140 (1 µM) on day 5; curve 3, undifferentiated, embryonal P19 cells. B, Changes in [Ca2+]i following stimulation by CCh. Curve 1, P19 neurons at day 8 of differentiation; curve 2, undifferentiated P19 cells; curve 3, P19 cells on day 8 following pretreatment with HOE-140 (1 µM) on day 5. curve 4, P19 neurons at day 8 of differentiation pretreated for 30 min with 180 ng/ml thapsigargin in order to deplete internal calcium stores. C, recovery of BK-induced calcium response following washout of HOE-140. Curve 1, changes in [Ca2+]i of P19 cells at day 8 of neuronal differentiation following addition of BK (1 µM). Curve 2, recovery of the calcium response induced by BK following addition of HOE-140 and wash-out for 75 min. Arrows indicate time points of BK injection. D, representative images of calcium responses following stimulation by BK and CCh: Left panels, embryonal carcinoma cells; middle panels, P19 neurons on day 7 of differentiation. Right panels, P19 cells on day 7 of differentiation, which had been pretreated with HOE-140 on day 5. -, unstimulated cells; +, maximal response observed following stimulation. In upper and lower panels, BK (1 µM) or CCh (1.5 mM) was used for stimulation of the cells. E, the average maximal increases in [Ca2+]i ± S.D. following BK or CCh addition to a control P19 cell population on day 7 were compared with BK- or CCh-stimulated P19 cells that had been pretreated on day 5 with HOE-140 (1 µm) or with the control peptide Lys-des-Arg9-Leu8-BK (leu 8) (1 µM). The data shown are mean values of two independent experiments that at least were performed in triplicate.

 
B2BKRs are expressed in the growth cones of differentiating PC12 pheochromocytoma cells and also in brain regions such as dorsal root ganglia. B2BKR expression was up-regulated following nerve injury or treating of primary cultures with neurotrophins (45), implicating a crucial role of B2BKRs in the repair and differentiation processes of neurons. A possible function of the B2BKR in neuronal development was found by Schelb et al. (12), who observed the postnatal expression of B2BKRs in sympathetically innervated tissues that modulate neurotransmitter release. The regulation of transmitter release, considered to be crucial in the development of the nervous system, was studied in PC12 cells and chromaffin tissue (5–8). Modulation of the B2BKR is implicated in neurodegeneration occurring during aging and Alzheimer disease (46). The suggestion that dysregulation of B2BKR function and expression could be implicated in the etiology of neurodegeneration and brain diseases is supported by the work of Arganaraz et al. (47) who detected an alteration of B2BKR expression in the hippocampus of a rat model of epilepsy.

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 {beta}-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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  		FIG. 6.
Quantification (day 8) of M1, M2, and M3 mAChR mRNA and differential mRNA transcription levels of M3 mAChR during neuronal differentiation of P19 cells. A, for quantification of differential expression of M1, M2, and M3 mAChR mRNA, total RNAs from P19 neurons at day 8 and P19 cells which had been pretreated on day 5 with HOE-140 (1 µM) were prepared, and M1–M3 mAChR mRNA transcription was determined by semi-quantitative RT-PCR as described under "Experimental Procedures." Details on primers and the size of the PCR products are given in Table I. Variations in cDNA concentration were normalized with the internal control {beta}-actin (Act). Control experiments were performed in the absence of the cDNA template. The intensities of the bands of the PCR products following ethidium bromide staining were determined by densitometry. B, quantification of mRNA transcription is given as x-fold increase over threshold background levels. The figure represents the results of three independent experiments that were performed in triplicate (*, p < 0.05). C, total RNAs from undifferentiated P19 cells (UN) and cells on days 4, 6, 8 and 10 of differentiation were prepared, and M3 mAChR (upper panel) and {beta}-actin (lower panel) mRNA transcription was analyzed by RT-PCR.

 
As B2BKRs could be expressed by glial cells that appear during RA-induced differentiation, it was important to show that B2BKRs are expressed by P19 neurons. Double-staining immunofluorescence studies showed that during neuronal differentiation in vitro, B2BKRs are expressed by neurons and the BK-induced effect is not due to activation of B2BKRs expressed on glial cells, which may be present in the differentiating culture (Fig. 2) (52).

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 {gamma}-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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  		FIG. 7.
HOE-140 treatment on EBs (48 h). P19 undifferentiated cells were plated in Petri dishes at a concentration of 105 cells/ml defined medium with 1 µM of RA or 1 µM each of RA and B2BKR antagonist HOE-140 (100 nM to 1 µM). After 48 h, 30 randomly chosen areas of each cell culture were photographed using a digital camera coupled to the microscope, and the size of EBs was measured and compared with EBs of cell cultures in the absence of HOE-140. A, the figure shows the results of a representative experiment. The left panel shows cells without HOE-140 treatment, and the right panel shows cells treated with 1 µM HOE-140. The scale bar indicates a size of 500 µm. B, the sizes of EBs were measured and compared with the control that had not been pretreated with HOE-140. p values of concentrations of 10 nM to 1 µM versus control were smaller than 0.05 using the unpaired Student's t test. C shows the percentages of relative sizes of untreated EBs compared with the sizes of EBs formed in the presence of HOE-140. The data are plotted as mean values ± S.E.

 
Our data indicate that B2BKR function is needed for termination of neuronal differentiation and the formation of functional synapses. Control experiments were carried out at identical conditions in the presence of the kinin B1 receptor antagonist Lys-des-Arg9-Leu8-BK, which reportedly does not affect B2BKR activity in order to verify HOE-140 specificity in inhibiting neuronal differentiation and to exclude the possibility of unspecific effects caused by micromolar peptide concentrations. Pretreatment of P19 cultures on day 5 with Lys-des-Arg9-Leu8-BK did not affect differentiation as determined by measuring mAChR activity on day 7 (Fig. 5E). For obtaining complete inhibition of B2BKR function, HOE-140 has been employed at similar concentrations in other in vitro assays, such as reversion of kinin-induced anion secretion in porcine ileum (58).

Previously published work supports the hypothesis of B2BKR participation in neuronal differentiation. In PC12 cells, BK signaling resulted in translocation of protein kinase C{epsilon}, 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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  		FIG. 8.
P19 cells as model for in vitro differentiation. The pattern of neurofilament expression in vitro mimics the normal pattern of neurofilament expression in vivo. NFL and NFM are expressed early in neuronal differentiation corresponding to day 2 or 3 following induction by RA (52). NFH (NF-200) is expressed later in neuronal differentiation, beginning on day 5 in P19 neurons and peaking on day 8 (data not shown). B2BKR mRNA and protein expression are present in undifferentiated cells as well as in EBs. During end-terminal differentiation of P19 neurons, B2BKR expression increases and reaches maximal levels together with NF-200 on day 8 following addition of RA. Inhibition of B2BKR activity on early neuronal differentiation was studied by addition of HOE-140 on day 0 and measuring the size of EBs after day 2. For evaluation of the effects of negative modulation of B2BKR activity on end-terminal differentiation of P19 neurons, the inhibitor HOE-140 was added on day 5, and mAChR mRNA transcription and activity were quantified on days 7 and 8. X indicates the time point of HOE-140 addition to the cultures. M, mitotic cells; PM, post-mitotic cells in neuronal maturation.

 
Despite the differential expression of various neuropeptide, neurotransmitter, and neurotrophin receptors (NTRs), such as the endothelin-B receptor, glutamate receptors, and the p75 NTR in differentiating P19 cells (24, 66, 67), none has been shown directly to be essential during their neuronal differentiation. Fig. 8 shows a scheme that illustrates the results reported herein. BK is generated by cleaving of kininogen precursors and is liberated into the cell culture medium, where it acts on B2BKRs, implicating that P19 cells use an autocrine loop system for triggering neuronal differentiation. As the half-time of BK in plasma is only 15 s (54), this peptide can activate receptors at specific time points without causing long-lasting receptor desensitization. Inhibition of B2BKR action at the initial stage of differentiation negatively affects formation of EBs as prerequisites for later differentiation. Blocking of B2BKR activity during later neuronal differentiation results in a decrease in muscarinic receptor activity and M1–M3 subtype expression. Thus, it is proposed that B2BKR-induced modulation of gene expression, at least of mAChR M1–M3 subtypes, is important for the acquisition of a specific neuronal phenotype.

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.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** 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. Back


   ACKNOWLEDGMENTS
 
We thank Prof. Antonio C. Cassola (Department of Physiology, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil) for allowing us to use the confocal microscope. We thank Prof. H. C. Schaller (Center for Molecular Neurobiology, University of Hamburg, Germany) for giving us the P19 cell line. The Center for Applied Toxicology (CAT-CEPID), Butantan Institute, São Paulo, Brazil, is acknowledged for performing DNA sequencing. Arthur A. Nery and Cleber A. Trujillo are acknowledged for helping us with graphical artwork.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Marceau, F., Hess, J. F., and Bachvarov, D. R. (1998) Pharmacol. Rev. 50, 357-386[Abstract/Free Full Text]
  2. Calixto, J. B., Cabrini, D. A., Ferreira, J., and Campos, M. M. (2000) Pain 87, 1-5[CrossRef][Medline] [Order article via Infotrieve]
  3. Bhoola, K. D., Elson, C. J., and Dieppe, P. A. (1992) Br. J. Rheumatol. 31, 509-518[Abstract/Free Full Text]
  4. Iwadate, H., Kudo, M., and Kizuki, K. (2002) Can. J. Physiol. Pharmacol. 80, 245-248[CrossRef][Medline] [Order article via Infotrieve]
  5. Kansui, Y., Fujii, K., Goto, K., and Abe, I. (2002) Hypertension 39, 29-34[Abstract/Free Full Text]
  6. Kurz, T., Tolg, R., and Richardt, G. (1997) J. Mol. Cell. Cardiol. 29, 2561-2569[CrossRef][Medline] [Order article via Infotrieve]
  7. Eurin, J., Barthelemy, C., Masson, F., Soualmia, H., Sarfati, E., and Carayon, A. (2002) Neuropeptides 36, 257-262[CrossRef][Medline] [Order article via Infotrieve]
  8. Dendorfer, A., Hauser, W., Falias, D., and Dominiak, P. (1996) Pfluegers Arch. 432, R99-R106[Medline] [Order article via Infotrieve]
  9. Thayer, S. A., Perney, T. M., and Miller, R. J. (1988) J. Neurosci. 8, 4089-4097[Abstract]
  10. El-Dahr, S. S. (1997) Microsc. Res. Tech. 39, 222-232[CrossRef][Medline] [Order article via Infotrieve]
  11. El-Dahr, S. S., Dipp, S., Meleg-Smith, S., Pinna-Parpaglia, P., and Madeddu, P. (2000) Pediatr. Nephrol. 14, 288-296[CrossRef][Medline] [Order article via Infotrieve]
  12. Schelb, V., Gobel, I., Khairallah, L., Zhou, H., Cox, S. L., Trendelenburg, A. U., Hein, L., and Starke, K. (2001) Naunyn-Schmiedeberg's Arch. Pharmacol. 364, 359-571[CrossRef][Medline] [Order article via Infotrieve]
  13. Ma, Q. P., and Heavens, R. (2001) Neuroreport 12, 2311-2314[CrossRef][Medline] [Order article via Infotrieve]
  14. Kozlowski, M. R., Rosser, M. P., and Hall, E. (1988) Neuropeptides 12, 207-211[CrossRef][Medline] [Order article via Infotrieve]
  15. Bush, A. B., Borden, L. A., Greene, L. A., and Maxfield, F. R. (1991) J. Neurochem. 57, 562-574[Medline] [Order article via Infotrieve]
  16. Tarnok, A. (1997) Cytometry 27, 65-70[CrossRef][Medline] [Order article via Infotrieve]
  17. Stephens, G. J., Cholewinski, A. J., Wilkin, G. P., and Djamgoz, M. B. (1993) Glia 9, 269-279[CrossRef][Medline] [Order article via Infotrieve]
  18. Yano, K., Higashida, H., Inoue, R., and Nozawa, Y. (1984) J. Biol. Chem. 259, 10201-10207[Abstract/Free Full Text]
  19. Chau, L. Y., Lin, T. A., Chang, W. T., Chen, C. H., Shue, M. J., Hsu, Y. S., Hu, C. Y., Tsai, W. H., and Sun, G. Y. (1993) J. Neurochem. 60, 454-460[Medline] [Order article via Infotrieve]
  20. Reetz, G., and Reiser, G. (1994) J. Neurochem. 62, 890-897[Medline] [Order article via Infotrieve]
  21. McBurney, M. W., Jones-Villeneuve, E. M., Edwards, M. K., and Anderson, P. J. (1982) Nature 299, 165-167[CrossRef][Medline] [Order article via Infotrieve]
  22. Jones-Villeneuve, E. M., McBurney, M. W., Rogers, K. A., and Kalnins, V. I. (1982) J. Cell Biol. 94, 253-262[Abstract/Free Full Text]
  23. Niemann, S., and Schaller, H. C. (1996) Neurosci. Lett. 207, 49-52[CrossRef][Medline] [Order article via Infotrieve]
  24. Monge, J. C., Stewart, D. J., and Cernacek, P. (1995) J. Biol. Chem. 270, 15385-15390[Abstract/Free Full Text]
  25. Staines, W. A., Morassutti, D. J., Reuhl, K. R., Ally, A. I., and McBurney, M. W. (1994) Neuroscience 58, 735-751[CrossRef][Medline] [Order article via Infotrieve]
  26. Pyle, S. J., Roberts, K. G., and Reuhl, K. R. (2001) Dev. Brain Res. 132, 103-106[CrossRef][Medline] [Order article via Infotrieve]
  27. Cardoso, C. C., Garrett, T., Cayla, C., Meneton, P., Pesquero, J. B., and Bader, M. (2004) Biol. Chem. 385, 295-301[CrossRef][Medline] [Order article via Infotrieve]
  28. Livak, K. J., and Schmittgen, T. D. (2001) Methods 25, 402-408[CrossRef][Medline] [Order article via Infotrieve]
  29. Laemmli, U. K. (1970) Nature 227, 680-685[CrossRef][Medline] [Order article via Infotrieve]
  30. Casarini, D. E., Boim, M. A., Stella, R. C., and Schor, N. (1999) Am. J. Physiol. 277, F66-F74[Medline] [Order article via Infotrieve]
  31. Hallett, M. B., Dormer, R. L., and Campbell, A. K. (1990) in Peptide Hormone Action: A Practical Approach (Siddle, K., and Hutton, J. C., eds) pp. 115-150, Oxford University Press, New York
  32. Hock, F. J., Wirth, K., Albus, U., Linz, W., Alpermann, H. G., Anagnostopoulos, H., Henke, S., Breipohl, G., Konig, W., Knolle, J., and Scholkens, B. A. (1991) Br. J. Pharmacol. 103, 774-777
  33. Regoli, D., Nsa Allogho, S., Rizzi, A., and Gobeil, F. J. (1998) Eur. J. Pharmacol. 348, 1-10[CrossRef][Medline] [Order article via Infotrieve]
  34. Yoshikawa, K., Aizawa, T., and Hayashi, Y. (1992) Nature 359, 64-67[CrossRef][Medline] [Order article via Infotrieve]
  35. Parnas, D., Heldman, E., Branski, L., Feinstein, N., and Linial, M. (1998) J. Mol. Neurosci. 10, 17-29[Medline] [Order article via Infotrieve]
  36. De Jaco, A., Augusti-Tocco, G., and Biagioni, S. (2002) J. Neurosci. Res. 70, 756-765[CrossRef][Medline] [Order article via Infotrieve]
  37. Irving, A. J., Collingridge, G. L., and Schofield, J. G. (1992) Cell Calcium 13, 293-301[CrossRef][Medline] [Order article via Infotrieve]
  38. Dixon, B. S., and Dennis, M. J. (1997) Kidney Int. 61, (suppl.) 514-517
  39. Wu, J., Akaike, T., Hayashida, K., Miyamoto, Y., Nakagawa, T., Miyakawa, K., Muller-Esterl, W., and Maeda, H. (2002) Int. J. Cancer 98, 29-35[CrossRef][Medline] [Order article via Infotrieve]
  40. Taub, J. S., Guo, R., Leeb-Lundberg, L. M., Madden, J. F., and Daaka, Y. (2003) Cancer Res. 63, 2037-2041[Abstract/Free Full Text]
  41. Drube, S., and Liebmann, C. (2000) Br. J. Pharmacol. 131, 1553-1560[CrossRef][Medline] [Order article via Infotrieve]
  42. Mahabeer, R., Naidoo, S., and Raidoo, D. M. (2000) Metab. Brain Dis. 15, 325-335[CrossRef][Medline] [Order article via Infotrieve]
  43. Lee, S. D., Lee, B. D., Kim, Y., Suh, P. G., and Ryu, S. H. (2002) Neurosci. Lett. 294, 130-132
  44. Baptista, H. A., Avellar, M. C., Araujo, R. C., Pesquero, J. L., Schanstra, J. P., Bascands, J. L., Esteve, J. P., Paiva, A. C., Bader, M., and Pesquero, J. B. (2002) Mol. Pharmacol. 62, 1344-1355[Abstract/Free Full Text]
  45. Lee, Y. J., Zachrisson, O., Tonge, D. A., and McNaughton, P. A. (2002) Mol. Cell Neurosci. 19, 186-200[CrossRef][Medline] [Order article via Infotrieve]
  46. Jong, Y. J., Dalemar, L. R., Seehra, K., and Baenziger, N. L. (2002) Int. Immunopharmacol. 2, 1833-1840[CrossRef][Medline] [Order article via Infotrieve]
  47. Arganaraz, G. A., Silva, J. A., Jr., Perosa, S. R., Pessoa, L. G., Carvalho, F. F., Bascands, J. L., Bader, M., da Silva Trindade, E., Amado, D., Cavalheiro, E. A., Pesquero, J. B., and da Graca Naffah-Mazzacoratti, M. (2004) Brain Res. 23, 114-125
  48. Jones-Villeneuve, E. M., Rudnicki, M. A., Harris, J. F., and McBurney, M. W. (1983) Mol. Cell. Biol. 12, 2271-2279
  49. McBurney, M. W. (1993) Int. J. Dev. Biol. 37, 135-140[Medline] [Order article via Infotrieve]
  50. Pyle, S. J., Roberts, K. G., and Reuhl, K. R. (2001) Develop. Brain Res. 132, 101-106
  51. Pizard, A., Blaukat, A., Michineau, S., Dikic, I., Müller-Esterl, W., Alhenc-Gelas, F., and Rajerson, R. M. (2001) Biochemistry 40, 15743-15751[CrossRef][Medline] [Order article via Infotrieve]
  52. McBurney, M. W., Reuhl, K. R., Ally, A. I., Nasipuri, S., Bell, J. C., and Craig, J. (1988) J. Neurosci. 8, 1063-1073[Abstract]
  53. Parnas, D., and Linial, M. (1995) Int. J. Dev. Neurosci. 13, 767-781[CrossRef][Medline] [Order article via Infotrieve]
  54. Morinelli, T. A., Meier, G. P., Webb, J. G., Jaffa, A. A., Privitera, P. J., and Margolius, H. S. (2001) Peptides 22, 2169-2174[CrossRef][Medline] [Order article via Infotrieve]
  55. Young, S. H., and Poo, M. M. (1983) Nature 305, 634-637[CrossRef][Medline] [Order article via Infotrieve]
  56. Baumgartner, M. K., Wei, J., and Aronstam, R. S. (1993) Brain Res. 72, 305-308
  57. Lin, P., Kusano, K., Zhang, Q., Felder, C. C., Geiger, P. M., and Mahan, L. C. (1996) J. Neurochem. 66, 233-242[Medline] [Order article via Infotrieve]
  58. Green, B. T., Calvin, A., O'Grady, S. M., and Brown, D. R. (2003) J. Pharmacol. Exp. Ther. 305, 733-739[Abstract/Free Full Text]
  59. Graness, A., Adomeit, A., Ludwig, B., Müller, W.-D., Kaufmann, R., and Liebmann, C. (1997) Biochem. J. 327, 147-154[Medline] [Order article via Infotrieve]
  60. Martin, A. K., Nahorski, S. R., and Willars, G. B. (1999) Br. J. Pharmacol. 126, 1559-1566[CrossRef][Medline] [Order article via Infotrieve]
  61. Fink, C. C., Slepchenko, B., Moraru, I. I., Watras, J., Schaff, J. C., and Loew, L. M. (2000) Biophys. J. 79, 163-183[Medline] [Order article via Infotrieve]
  62. Gomez, T. M., and Robles, E. (2003) Methods Enzymol. 361, 407-422[Medline] [Order article via Infotrieve]
  63. Purkiss, J. R., Nahorski, S. R., and Willars, G. B. (1995) J. Neurochem. 64, 1175-1182[Medline] [Order article via Infotrieve]
  64. Bush, A. B., Borden, L. A., Greene, L. A., and Maxfield, F. R. (1991) J. Neurochem. 562-574
  65. Reber, B. F. X., and Schindelholz, B. (1996) Pflugers Arch. 432, 893-903[CrossRef][Medline] [Order article via Infotrieve]
  66. Canzoniero, L. M., Sensi, S. L., Turetsky, D. M., Finley, M. F., Choi, D. W., and Huettner, J. E. (1996) J. Neurosci. Res. 45, 226-236[CrossRef][Medline] [Order article via Infotrieve]
  67. Burke, M. A., and Bothwell, M. (2003) J. Neurobiol. 55, 191-203[CrossRef][Medline] [Order article via Infotrieve]

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R. L. da Silva, R. R. Resende, and H. Ulrich
Genomic Physiology: Alternative splicing of P2X6 receptors in developing mouse brain and during in vitro neuronal differentiation
Exp Physiol, January 1, 2007; 92(1): 139 - 145.
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