BNP and NPR-A are expressed in vivo in adult mouse TG

Gene expression of BNP and its receptor NPR-A investigated by RT-PCR indicated weakly positive BNP and strongly positive NPR-A bands of the expected size (199 bp and 200 bp respectively; Figure 1 A). We next examined with Western blot analysis if NPR-A protein was synthesized. The NPR-A antibody recognized a single band of the expected size (approximately 140 kDa) in mouse TG homogenates, corresponding to the NPR-A protein. Conversely, BNP expression was below detection. Thus, BNP expression was evaluated by ELISA assay. Mouse BNP concentrations in tissue extracts from entire TG and in the serum were 1.12 ± 0.34 pg/mg protein and 185.2 ± 47 pg/ml, respectively (n=6 mice). Western blot analysis confirmed that NPR-A receptor was synthesized both in adult (P30) and in younger (P12) animal TG (Figure 1 B). Thus, the properties of this peptide could be investigated with younger tissues as well, a convenient preparation for primary culture studies.

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Figure 1. Expression of BNP and NPR-A in adult mouse TG in vivo.

A, PCR products (amplified with 40 cycles) identified by ethidium bromide staining show RT-PCR amplification of BNP and NPR-A mRNA (+RT), as well as of the housekeeping gene GAPDH. Negative control reactions for retrotranscription (-RT) do not show any bands. B, Chemiluminescence image of Western blot showing protein expression of NPR-A in TG from P30 and P12 mice. β-Tubulin III was used as a loading control. C, Example of confocal microscopy images showing the widespread colocalization of immunostaining for NPR-A (red) and β-tubulin III (green). Cell nuclei are visualized with DAPI staining (blue). Scale bar, 100 µm. D, Representative confocal images of longitudinal sections of adult TG. Immunohistochemistry reveals BNP-immunoreactive granules around few nuclei (DAPI). Among these cells, most possess non-neuronal morphology (left panel); neuron-like cells are rarely immunostained (right panel). Scale bar, 30 µm.

https://doi.org/10.1371/journal.pone.0081138.g001

The distribution of NPR-A and BNP in mouse TG was further examined by immunohistochemistry. We found that NPR-A was expressed in most neurons, as shown by co-localization with β-tubulin III (Figure 1 C). NPR-A immunoreactivity could not be detected in other cell types. Further observations (Figure S1 A) confirmed that the NPR-A staining appeared colocalized with TRPV1 and P2X3 immunopositive neurons. Unlike the data concerning its receptor, BNP immunopositive cells were few and scattered elements, in which the peptide staining appeared as small perinuclear granules (0.9 ± 0.4% of total ganglion cells, n=3) (Figure 1 D). The few BNP-immunoreactive cells seldom showed neuron-like morphology.

Characterization of BNP and NPR-A in TG primary cultures

In order to examine the characteristics of BNP effects on TG cells, we used TG primary cultures as previously reported [18,39]. Consistent with our results from tissue sections, double immunostaining for NPR-A and the neuronal marker β-tubulin III showed that in vitro the receptor was expressed exclusively by neurons (Figure 2 A). Figure 2 B summarizes the distribution of NPR-A immunoreactivity in relation to the neuronal cell body size which was the highest in neurons with somatic diameter between 12 and 25 µm, consistent with the higher prevalence of small and medium sized neurons in TG [18]. Systematic cell counting indicated that the vast majority of β-tubulin III-positive cells expressed NPR-A (91.3 ± 1.1%, n=10), confirming the extensive colocalization of the two proteins already observed in vivo. Western blot analysis with biotinylation method validated the expression and membrane localization of NPR-A (Figure 2 C).

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Figure 2. BNP and NPR-A are expressed in mouse TG cultures.

A, Immunocytochemical analysis of NPR-A (red) and β-tubulin (green) in mouse TG culture. Nuclei are visualized with DAPI (blue). Merge image (right) indicates extensive co-staining. Scale bar, 30 µm. B, Somatic size distribution of NPR-A-positive TG neurons. Data represent 119 NPR-A-positive neurons from 4 independent experiments. C, Example of Western immunoblotting (representative of three experiments) showing the surface (obtained after membrane biotinylation) and total expression of NPR-A in TG culture. β-Actin was used as loadingcontrol of the total extract. TfR is the marker for correct biotinylation. D, Immunostaining for BNP (green) is mostly restricted to few cells with non-neuronal morphology that display perinuclear immunoreactive granules (left panel). More rarely, some neuron-like cells are also stained for BNP with granules extending along their processes (right panel). Nuclei are visualized with DAPI staining (blue). Scale bar, 30 µm.

https://doi.org/10.1371/journal.pone.0081138.g002

Immunocytochemistry revealed the scant presence of BNP-positive cells also in TG primary cultures, as they accounted for only 2.0 ± 0.2% of the total cell population in our cultures (n=12). The morphology of these cells appeared prevalently non-neuronal (Figure 2 D, left), although rare neuron-like cells were observed as well (Figure 2 D, right). We also estimated (with ELISA assay) the concentration of BNP in primary cultures and in their medium that was found to be 1.1±0.4 pg/mg and 4.9±2.6 ng/ml, respectively (n=8 mice).

Functional assays of NPR-A

We sought to understand whether the large expression of membrane bound NPR-A was actually indicative of a functional receptor system. To this end, since activation of NPR-A by ANP and BNP has been shown to increase intracellular cGMP production [40], we investigated if application of BNP to mouse TG cultures could increase intracellular cGMP production, as determined by ELISA. Figure 3 A shows that application of 100 or 500 ng/ml BNP (compared to the vehicle control treatment) increase the mean cGMP levels in a concentration- and time-related fashion. In fact, the cGMP rise was already detectable after 5 min and reached its peak after 30 min with a late (1 h) decline. The greatest increment was observed after 30 min application of 500 ng/ml BNP. Even 100 ng/ml BNP was sufficient to activate the NPR-A mediated pathways by significantly increasing cGMP by almost 2-fold compared to basal levels. This effect was prevented by the application of 500 nM anantin, a specific antagonist of NPR-A [41,42], that completely inhibited the elevation of cGMP levels. A lower anantin concentration (100 nM) failed to produce significant antagonism of BNP-elicited rise in cGMP (Figure 3 A). It is noteworthy that anantin (500 nM) per se significantly decreased the basal level of cGMP.

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Figure 3. cGMP production by mouse TG cultures.

A, ELISA-based quantification of intracellular cGMP in primary cultures from P12 mice. Cultures were treated with BNP (100 or 500 ng/ml for 5, 10 or 30 min) and/or anantin (100 and 500 nM, 30 min), or treated with vehicle (indicated as 0). Note large rise in cGMP that declines at 60 min; anantin alone (500 nM) decreases basal cGMP. Data were normalized to the total protein content in each sample and are presented as mean value ± SEM (n=3). *p≤0.05. B (top), example of western blot of Akt phosphorylation induced by BNP (500 ng/ml) applied alone or together with anantin (100 and 500 nM) for various times as indicated. Bottom histograms quantify these data from at least 3 experiments, demonstrating early and transient increment in P-Akt expression.

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One important intracellular effect of NPR-A activation is phosphorylation of Akt that occurs downstream of cGMP synthesis [43]. We tested whether this phenomenon could be also observed in TG cultures. Figure 3 B shows that after 10 or 30 min application of 500 ng/ml BNP, there was a significant increase in Akt phosphorylation, an effect fully blocked by 500 nM anantin, yet insensitive to 100 nM antagonist concentration. It was of interest that the increment in Akt phosphorylation was not sustained as it declined at 30 min and disappeared 24 h later (Figure 3 B).

These experiments, thus, demonstrated that functional NPR-A receptors were found in TG cultures despite the poor expression of BNP. We wondered if activation of these receptors could be translated into changes in the activity of ligand-gated neuronal channels considered to be important transducers of nociceptive signals. For this purpose, we studied how BNP application could affect electrophysiological responses mediated by P2X3 or TRPV1 channels.

Effect of BNP on P2X3 and TRPV1 receptors

Patch clamp experiments were run to test whether short or long term application of 100 ng/ml BNP could change responses evoked by α,β-meATP (10 µM) or capsaicin (1 µM) to activate P2X3 or TRPV1 receptors, respectively [18]. As a reference, we also tested the inward, Cl^- mediated currents generated by GABA (10 µM) via activation of GABA_A receptors [44,45]. Only one concentration of BNP (100 ng/ml) was used for electrophysiological experiments, since we had shown it to be sufficient to activate NPR-A-mediated pathways and to increase significantly cGMP levels (Figure 3 A); the same BNP concentration was also used in previous studies, showing its effectiveness on DRG neurons [25].

Figure 4 A shows that continuous superfusion with BNP (100 ng/ml for 10 min) did not change sample responses to these three ligands. Furthermore, BNP did not change cell input resistance (528±50 MΩ in control versus 552±57 MΩ in the presence of BNP; n=28) or baseline current (84±15 versus 79±11 pA before and after BNP, respectively; n= 28). Preincubating cultures for 2 h with 100 ng/ml BNP also failed to alter the responses mediated by P2X3, GABA_A or TRPV1 receptors (Figure 4 B). Nonetheless, 24 h application of 100 ng/ml BNP significantly and selectively depressed the currents evoked by 1 µM capsaicin (Figure 5 A, see arrows). This effect was also observed for other capsaicin concentrations (0.1 µM, 0.5 and 5 µM; see Figure 5 B) as the dose/response plot was shifted downwards after chronic application of BNP.

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Figure 4. Ten min or 2 h BNP application does not affect capsaicin-, α,β-meATP- or GABA-mediated responses.

A, Representative traces of currents induced by pulse application of α,β-meATP (10 µM, 2 s), capsaicin (1 µM, 3 s), or GABA (10 µM, 2 s) to TG neurons in control conditions or after application of BNP (100 ng/ml, 10 min) to the same cells. Histograms show average current density values of P2X3, TRPV1 or GABA-mediated responses (n=12, 12, 11, respectively). B, Histograms show average current density values of P2X3, TRPV1 or GABA-mediated responses in control conditions (n=52, 73, 123, respectively) or after 2 h application of 100 ng/ml BNP (n=42, 53, 119).

https://doi.org/10.1371/journal.pone.0081138.g004

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Figure 5. Changes in current responses produced by capsaicin, α,β-meATP or GABA following 24 h BNP application.

A, Histograms show average current density values of TRPV1-mediated responses induced by 1 µM capsaicin in TG neurons under control conditions or after 24 h application of BNP (100 ng/ml); n=151 and 109 for control and after application of BNP, respectively; *p= 0.00014. Inset shows an example of superimposed currents of one neuron in control and after BNP treatment. B, Dose-response curves for capsaicin mediated currents in control and after 24 h BNP treatment (100 ng/ml); *p<0.035; n≥ 15 for each agonist concentration. C, Histograms summarize average current density values of the responses to 1 µM capsaicin in control conditions, after 24 h application of 500 nM anantin, or anantin plus 100 ng/ml BNP (n=53, 50, 43, respectively). D, E, Histograms show average current density values of P2X3-mediated currents in control conditions and after 24 h application of BNP (100 ng/ml); n=74 and 76, respectively. Anantin alone significantly increases these values compared to control, an effect not reversed by co-application with BNP (n=69, 63, 64, respectively); *p<0.0001. F, Dose-response curves for α,β-meATP mediated currents in control and after 24 h exposure to 500 nM anantin ; *p<0.035; n≥14 for each agonist concentration. G, Western blot analysis of TRPV1 or P2X3 receptor expression 24 h after applying BNP (100 ng/ml) or anantin (500 nM); data are from 3 experiments. H, I, Histograms show no change in GABA-mediated responses after 24 h application of BNP (100 ng/ml; n=25 and 27, respectively). Likewise, there is no change after application of anantin alone, or with BNP (n=73, 73, or 86, respectively).

https://doi.org/10.1371/journal.pone.0081138.g005

We next explored whether this slow action by BNP could be attributed to the consequence of NPR-A receptor activation followed by a rather slow series of events culminating in TRPV1 channel modulation. Should this hypothesis be correct, the effects of BNP ought to be suppressed by the selective antagonist of NPR-A anantin [32,41,42]. Thus, we first validated the use of anantin, that, when applied alone at 500 nM (to ensure a complete blockage of NPR-A; Figure 3 A, B), did not change control responses to capsaicin α,β-meATP. Figure S1 B shows that preapplication of anantin (500 nM; 10 min) failed to change P2X3 receptor responses. Likewise, responses evoked by capsaicin (1 µM) were 11.9±4.3 pA/pF (n=6), a value not significantly different from the one observed after anantin (9.5±5.1 pA/pF, n=4), suggesting no direct channel modulation by anantin.

Figure 5 C shows that the inhibitory effect on TRPV1 receptors seen after 24 h application of BNP was fully prevented by concomitant administration of 500 nM anantin (that per se had no effect on capsaicin-induced responses even when chronically applied). No significant change in P2X3 receptor-mediated currents was present after 24 h BNP application (Figure 5 D). It was, however, interesting to observe that anantin (500 nM; 24 h) per se enhanced responses to α,β-meATP (10 µM), that were almost twice (196 %) their controls (Figure 5 E). This effect was detected at various agonist concentrations as the α,β-meATP dose-response curve was shifted leftwards without significant change in the maximum response amplitude (Figure 5 F). When anantin plus BNP were continuously co-applied for 24 h, the potentiation of the P2X3 receptor currents persisted (Figure 5 E). Figure 5 G shows that 24 h application of BNP (100 ng/ml) or anantin (500 nM) did not change the expression level of either TRPV1 or P2X3 receptors. Finally, we did not find any change in GABA-mediated currents after applying BNP, or anantin, or their combination (Figure 5 H, I).

Although short-term BNP and/or anantin application was sufficient to produce fast changes in cGMP levels or Akt phosphorylation, modulation of TRPV1 or P2X3 currents appeared only after 24 h of BNP and/or anantin exposure. Thus, we wondered if even a short BNP or anantin treatment could trigger a very delayed receptor modulation. For this, we applied BNP (100 ng/ml) or anantin (500 nM) for 1 h only and, after overnight wash, patch-clamping was performed. With this protocol, the effects of BNP and anantin on TRPV1 or P2X3 currents were no longer observed (Figure S2).

Preparations of mouse trigeminal ganglia and immunostaining

Primary cultures of P12 mouse trigeminal ganglia were prepared as described previously [18,39] and used 24 h after plating.

For immunohistochemistry, mice were deeply anesthetized with intraperitoneal injection of urethane (0.3 ml of 1 g/ml; Sigma, Milan, Italy) and perfused transcardially with phosphate buffer solution followed by 4% paraformaldehyde. Trigeminal ganglia were removed, postfixed for 1 h at room temperature and cryoprotected overnight in 30% sucrose at 4°C. Each immunohistochemistry experiment was performed on an average of 5 cryostat-cut serial longitudinal slices (14 µm-thick) sampled every ~70 µm, and thus covering the entire ganglion. Samples were incubated in a blocking solution containing 5% bovine serum albumin, 1% fetal bovine serum and 0.1% Triton X-100 in phosphate saline buffer for 2 h at room temperature, and immunostained with primary (for 16 h at 4°C) and secondary antibodies (2 h at room temperature). Antibodies against NPR-A (1:1000; Abcam, Cambridge, UK), BNP (1:100; Phoenix Pharmaceuticals, Burlingame, CA, USA) and β-tubulin III (1:1000; Sigma) were used. The specificity of the NPR-A antibody used in this study has been previously validated [25]. Secondary antibodies conjugated with Alexa Fluor-488 or Alexa Fluor-594 were purchased from Invitrogen (1:500; Milan, Italy). Nuclei were counterstained with DAPI (Sigma). The scant immunostaining for BNP led us to check whether this phenomenon could be attributed to a fixation artifact. Thus, we cut, with a cryostat, freshly frozen ganglia (14 µm sections) and processed them for BNP immunoreactivity using the blocking solution indicated above. Even in this case, BNP signals were absent or very rare. Double immunofluorescence experiments were performed using Zenon Technology: the anti-NPR-A antibody was labeled with Zenon Alexa-594 rabbit IgG labeling reagent as per manufacturers’ instructions (Invitrogen). Images from whole ganglion sections were visualized with Leica confocal microscope (Leica TCS SP2, Wetzlar, Germany) or a Zeiss Axioskop fluorescence microscope (Zurich, Switzerland). Similar procedures were used for fluorescence immunostaining of cultured mouse TG neurons [18,58]. MetaMorph software (Molecular Devices, Downingtown, PA, USA) was employed for data analysis.

Western blot

Western blot analysis was performed according to the methods previously reported [58,59]. Cells were lysed in buffer solution (2% n-octyl-beta-D-glucopyranoside, contaning 1% Nonidet P-40, 10 mM Tris pH 7.5, 150 mM NaCl plus protease inhibitors mixture; Complete, Roche Applied Science, Basel, Switzerland) and immunoblotted with rabbit anti-NPR-A (1:1000, Abcam), anti-β-tubulin III (1:2000; Sigma) or anti-β-actin (1:3000, Sigma) antibodies. For Akt phosphorylation assays, cells were lysed in phosphate buffer solution containing 0.1% SDS, 1% sodium deoxycholate, and 1% triton X-100, and immunoblotted with anti-phospho-Akt Ser473 (1:1000; Cell Signaling, Danvers, MA, USA), anti-Akt (1:1000; Cell Signaling) or anti-β-actin-HRP (1:3000; Sigma). Signals were detected with the enhanced chemiluminescence light system ECL (Amersham Biosciences, Piscataway, NJ, USA) and recorded by the digital imaging system Alliance 4.7 (UVITEC, Cambridge, UK). Quantification of the optical density of the bands was performed with ImageJ software plug-in (Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/, 1997-2012).

Biotinylation of surface expressed receptors

For biotinylation experiments, cells were incubated with 1 mg/ml EZ-Sulfo-NHS-LC-biotin (Pierce, Rockford, IL, USA) as previously described [39,60]. Pull-down of biotinylated proteins was obtained with Streptavidin agarose resin (Pierce) for 2 h at 4°C according to the manufacturer’s instructions. Samples were processed for Western immunoblotting using antibodies against the NPR-A receptor (1:1000). Biotinylation experiments resulted to be free of intracellular protein contaminants (as shown by lack of signal for β-actin). Positive control for biotinylation assay was obtained by checking the surface expression of the transferrin receptor detected with an antibody purchased from Santa Cruz Biotechnology (1:1000, Heidelberg, Germany). For control of correct gel loading, the expression of β-actin in the intracellular fraction was checked.

RNA isolation, reverse transcription and RT-PCR

Total RNA was extracted from mouse intact TG tissue samples using Trizol reagent (Invitrogen) according to the manufacturer’s instruction. All RNA samples were subjected to DNase I treatment (Ambion, Monza, Italy). A total of 1 μg of RNA was subjected to retrotranscription using iScript cDNA synthesis kit (BioRad, Hercules, CA, USA) and RT–PCR was carried out using SYBR green fluorescence dye (2× iQ5 SYBR Green supermix, BioRad) as described previously [61]. Specific primer sets for NPR-A [62], BNP [63], GAPDH [64] and β-actin [65] were previously described.

Assay for intracellular cGMP production in TG cultures

Quantification of BNP-induced intracellular cGMP production in mouse TG cultures was performed using a direct ELISA kit (MBL International Corporation, Woburn, MA, USA) following the instructions of the manufacturer. Briefly, cultures were lysed in 0.1 M HCl and subjected to determination of intracellular cGMP concentration. The protein concentrations in cell lysates were determined by the BCA method (Sigma). Intracellular cGMP concentrations were expressed as pmol of cGMP per µg of protein. Three independent experimental replicates were generated from three different batches of mouse TG cultures.

Serum sampling and ELISA assay for BNP

Blood was sampled from the retro-orbital sinus of mice, immediately transferred to chilled microtubes on ice for 30 min and centrifuged at 10,000 g at 4°C for 10 min. Culture medium was collected from TG cultures 24 h after plating and centrifuged at 10,000 g at 4°C for 10 min. From the same cultures, cells were lysed in 0.1 M HCl and centrifuged at 10,000 g at 4°C for 10 min. Protein concentrations in cell lysates were determined by the BCA method (Sigma). Tissue and medium samples were immediately used for ELISA assay to determine BNP concentrations following the instructions of the manufacturer (Abnova, Hidelberg, Germany). All samples were run in triplicate and values averaged.

Electrophysiology and data analysis

Electrophysiology experiments were performed according to the methods previously reported [17,18]. Briefly, after 24 h in culture, TG neurons were superfused continuously (3 mL/min) with physiological solution containing (in mM): 152 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, and 10 HEPES (pH adjusted to 7.4 with NaOH). Cells were patch-clamped in the whole-cell configuration using pipettes with a resistance of 3-4 Mʊ when filled with the following solution (in mM): 140 KCl, 0.5 CaCl₂, 2 MgCl₂, 2 Mg₂ATP₃, 2 GTP, 10 HEPES, and 10 EGTA (pH adjusted to 7.2 with KOH). Recordings were performed from small and medium size neurons that mostly express TRPV1 and/or P2X3 receptors [17,18]. Cells were held at -65 mV; membrane currents were filtered at 1 kHz and acquired by means of a DigiData 1200 interface and pClamp 8.2 software (Molecular Devices, Sunnyvale, CA, USA). To obtain stable and reproducible P2X3 receptor currents, its synthetic agonist α,β-methylene-adenosine-5’-triphosphate (α,β-meATP; Sigma) at the concentration of 10 µM that elicits near-maximal responses [58,64,66] was applied (for 2 s) using a fast superfusion system (Rapid Solution Changer RSC-200; BioLogic Science Instruments, Claix, France). α,β-meATP at 1, 3, 5, 30 and 100 µM concentrations was additionally studied to obtain dose-response curves in control and experimental conditions. In line with previous reports [17,58], capsaicin (Sigma) was applied at the concentration of 1 µM (3 s) to evoke reproducible inward currents. Additionally concentrations 0.1, 0.5, 5 and 10 µM were included to obtain capsaicin dose-response curves under control conditions and after the treatment. GABA (10 µM) was applied with 2 s pulses [45]. Peak current amplitudes were divided by the cell slow capacitance to express responses as current density values.

For patch clamp experiments, various protocols were used to test the effect of BNP on these ligand-gated channels. In a series of experiments, BNP was continuously superfused (starting 10 min prior to the agonist pulses) at 100 ng/ml concentration while agonist responses were repeatedly tested. In other experiments, TG cultures were incubated with BNP solution (100 ng/ml) for 2 or 24 h; after washing out the peptide, cells were immediately patch-clamped and tested for their responsiveness to the agonists. In the third type of the protocol BNP was applied for 1 h and, after wash out, cells were incubated for 23 h in the usual medium before the experiment. A similar approach was employed for the NPR-A antagonist anantin (500 nM) applied either alone or together with BNP.

Chemicals and reagents

Purified recombinant mouse BNP was purchased from Phoenix Pharmaceuticals; the specific NPR-A antagonist anantin was from US Biologicals (Salem, MA, USA). BNP and anantin were dissolved in distilled water as the datasheet suggested. BNP was applied at the dose of 100 ng/ml, as previously performed for sensory ganglia [25], and 500 ng/ml. Anantin was applied at the dose of 100 nM and 500 nM as previously reported [67].

To inhibit NPR-A receptor activity, we used anantin since, to the best of our knowledge, it is the only specific and selective NPR-A antagonist [41–43], devoid of agonistic activities [68], unlike commercially-available antagonists which are not selective on NPR subtypes [69–72], and which may also possess agonistic activities [73].

Statistical analysis

Data were collected from at least three independent experiments, and are expressed as mean ± standard error of the mean (SEM), where n indicates the number of independent experiments or the number of investigated cells, as indicated in figure legends. Statistical analysis was performed using the Student’s t-test or the Mann-Whitney rank sum test, after the software-directed choice of parametric or non-parametric data, respectively (Sigma Plot and Systat Software Inc., San Jose, CA, USA). A p value of ≤ 0.05 was accepted as indicative of a statistically significant difference.