Functional expression of bradykinin B1 and B2 receptors in neonatal rat trigeminal ganglion neurons

Bradykinin (BK) and its receptors (B1 and B2 receptors) play important roles in inflammatory nociception. However, the patterns of expression and physiological/pathological functions of B1 and B2 receptors in trigeminal ganglion (TG) neurons remain to be fully elucidated. We investigated the functional expression of BK receptors in rat TG neurons. We observed intense immunoreactivity of B2 receptors in TG neurons, while B1 receptors showed weak immunoreactivity. Expression of the B2 receptor colocalized with immunoreactivities against the pan-neuronal marker, neurofilament H, substance P, isolectin B4, and tropomyosin receptor kinase A antibodies. Both in the presence and absence of extracellular Ca2+ ([Ca2+]o), BK application increased the concentration of intracellular free Ca2+ ([Ca2+]i). The amplitudes of BK-induced [Ca2+]i increase in the absence of [Ca2+]o were significantly smaller than those in the presence of Ca2+. In the absence of [Ca2+]o, BK-induced [Ca2+]i increases were sensitive to B2 receptor antagonists, but not to a B1 receptor antagonist. However, B1 receptor agonist, Lys-[Des-Arg9]BK, transiently increased [Ca2+]i in primary cultured TG neurons, and these increases were sensitive to a B1 receptor antagonist in the presence of [Ca2+]o. These results indicated that B2 receptors were constitutively expressed and their activation induced the mobilization of [Ca2+]i from intracellular stores with partial Ca2+ influx by BK. Although constitutive B1 receptor expression could not be clearly observed immunohistochemically in the TG cryosection, cultured TG neurons functionally expressed B1 receptors, suggesting that both B1 and B2 receptors involve pathological and physiological nociceptive functions.


Introduction
Tissue damage results in an accumulation of endogenous chemical substances, such as bradykinin (BK), which are released by nociceptive afferents and/or non-neural cells in the injured area of the tissue (Julius and Basbaum, 2001;Basbaum et al., 2009). BK receptors, which are divided into two subtypes (B 1 and B 2 ), are plasma membrane G-protein-coupled receptors of the seven-transmembrane-domain family. The existence of B 1 and B 2 receptors has been confirmed by pharmacological and radioligand-binding studies, as well as by mRNA expression analyses, in a wide variety of cells (Hess et al., 1994;Pesquero et al., 1996;Hall, 1997).
Previous studies have indicated that B 2 receptors couple with the Gq protein. Activation of the Gq protein activates phospholipase C, which induces a number of intracellular second messenger systems, including 1, 2-diacylglycerol and inositol 1, 4, 5trisphosphate, which activates protein kinase C and mobilizes intracellular Ca 2+ , respectively (Walker et al., 1995;Tiwari et al., 2005).
BK-induced changes in the chemical environment surrounding axons cause peripheral sensitization, which is associated with inflammatory responses (Basbaum et al., 2009). Neuropathic pain is also involved in peripheral and central sensitization, which increases chronic pain states (Cervero and Laird, 1996;Scholz and Woolf, 2002;Ochoa, 2009). Injury to trigeminal ganglion (TG) neurons, which occasionally induces neuropathic pain, has been reported to be mediated by both B 1 and B 2 receptors in the orofacial area. Formalin-induced orofacial pain responses in rats are reduced by B 2 receptor inhibition (Chichorro et al., 2004). In addition, administration of B 1 and B 2 receptor antagonists delays the development of thermal hyperalgesia in the orofacial area, which is induced by constriction of the infraorbital nerve in rats and mice (Luiz et al., 2010). Thus, the functional role of BK receptors in TG neurons in physiological and pathological nociception has been well described by behavioral studies. However, the basic expression patterns of B 1 and B 2 receptors in TG neurons are still unclear and remain to be fully elucidated.
In the present study, we investigated the expression and localization, as well as physiological and pharmacological properties, of B 1 and B 2 receptors in primary cultured rat TG neurons.

Ethical Approval
All the animals used in our study were treated in accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences, which was approved by the Council of the Physiological Society of Japan and the American Physiological Society. In addition, the study followed the guidelines that were established by the National Institutes of Health (USA) regarding the care and use of animals for experimental procedures. This study was approved by the Animal Research Ethics Committee of Tokyo Dental College (approval No. 252502).

Measurement of [Ca 2+ ] i
Primary cultured TG cells were loaded for 90 min at 37 • C in Hank's solution containing 10 µM of fura-2 acetoxymethyl ester (Dojindo Laboratories, Kumamoto Japan) and 0.1% (w/v) pluronic acid F-127 (Life Technologies). Cultured TG cells were then rinsed with fresh Hank's solution and mounted on a microscope stage (Olympus Corporation, Tokyo, Japan). Fura-2 fluorescence emission was measured at 510 nm in response to alternating excitation wavelengths of 340 nm (F340) and 380 nm (F380) with an Aquacosmos system and software (Hamamatsu Photonics K.K., Shizuoka, Japan), which controls the excitation wavelength selector and intensified charge-coupled device camera system (Hamamatsu Photonics K.K.). [Ca 2+ ] i was measured as the fluorescence ratio of F340 and F380 (R F340/F380 ) and expressed as F/F 0 units. The R F340/F380 value (F) was normalized to the resting value (F 0 ).

Statistical and Offline Analysis
The data were expressed as the mean ± standard error (S.E.) or standard deviation of the mean of N observations, where N represents the number of independent experiments or cells, respectively. The Kruskal-Wallis test, Dunn's posthoc test, or Mann-Whitney U-test was used to determine the nonparametric statistical significance. P values less than 0.05 were considered significant. The statistical analysis was performed with GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA).
The dependence of the changes in [Ca 2+ ] i on each pharmacological agent was determined by fitting the data to the following function with Origin 8.5 (OriginLab Corporation, Northampton, MA, USA): (1) where K is the equilibrium binding constant, [x] o indicates the applied concentration of the pharmacological agents, and F/F 0int and F/F 0fin are the initial and final F/F 0 responses, respectively.

Immunolocalization of BK Receptors in TG Neurons
The cultured TG neurons showed positive immunoreactivity to a neuronal marker cocktail (Neuro-Chrom TM pan-neuronal marker), which contained mouse anti-NeuN, anti-MAP2, and anti-βIII tubulin antibodies (Figures 1A,D). Intense B 2 receptor immunoreactivity was observed in primary cultured TG neurons (Figure 1E), and it showed colocalization with the pan neuronal marker ( Figure 1F) in somata, dendrites, axons, and perinuclear regions. Weak but positive B 1 receptor immunoreactivity was also observed in primary cultured TG cells (Figure 1B), and the immunoreactivity colocalized with the pan neuronal marker ( Figure 1C).
In the TG cryosections, we could observe positive immunoreactivity against the neuronal marker cocktail (Figures 1G,J). These TG neurons in the cryosections showed positive immunoreactivity to the B 2 receptor antibody ( Figure 1K), showing colocalization with the pan neuronal marker ( Figure 1L) in somata, dendrites, axons, and perinuclear regions. However, the TG cryosections did not show B 1 receptor immunoreactivity (Figures 1H,I). Positive immunoreactivity was also observed with NF-H (an A-neuron marker; Figure 2A), SP (a peptidergic C-neuron marker; Figure 2D), IB4 (a nonpeptidergic C-neuron marker; Figure 2G), and high-affinity NGF receptor (TrkA; an NGF-responsive nociceptor marker; Figure 2J) antibodies. These immunoreactivities against NF-H, SP, IB4, and TrkA antibodies showed colocalization with those against the B 2 receptor antibodies (Figures 2B,C

BK-Induced [Ca 2+ ] i Increases in TG Neurons
We observed rapid and transient [Ca 2+ ] i increases in TG neurons following the administration of five different concentrations of BK (0.01, 0.1, 1.0, 10, and 100 nM) in the presence of external Ca 2+ (2.0 mM; Figure 3A). A semilogarithmic plot ( Figure 3B) illustrates F/F 0 values as a function of the applied BK concentrations, and the equilibrium-binding constant was the half-maximal 50% effective concentration (EC 50 ) of 1.0 nM.

HOE140, a B 2 Receptor Antagonist, Inhibited the BK-Induced [Ca 2+ ] i Increases in TG Neurons
We examined the BK-induced [Ca 2+ ] i responses in both the presence and absence of external Ca 2+ . The application of BK (1.0 nM) rapidly increased [Ca 2+ ] i to a peak F/F 0 value of 1.7 ± 0.03 F/F 0 units in the presence (2.0 mM) of external Ca 2+ and 1.4 ± 0.03 F/F 0 units in the absence (0 mM) of external Ca 2+ (Figures 4A,D). The amplitudes of the BK-induced [Ca 2+ ] i increases significantly differed between those in the presence and absence of extracellular Ca 2+ . In the absence of extracellular Ca 2+ , BK (1.0 nM)-induced [Ca 2+ ] i increases were significantly inhibited by a B 2 receptor antagonist (100 nM of HOE140) (Figures 4C,D) but not by a B 1 receptor antagonist (1.0 µM of R715) (Figures 4B,D).  (Figures 5A,B).

Pharmacological Identification of B 1 Receptors in TG Neurons
We investigated the [Ca 2+ ] i increases during the administration of Lys-[Des-Arg 9 ]BK, which is an endogenous, potent, and    (Figures 5E,F).

Discussion
The present study demonstrated the functional expression of BK receptors (B 1 and B 2 ) in TG neurons. B 2 receptors were present on axons and dendrites in A-neurons, nonpeptidergic C-neurons, peptidergic C-neurons, and NGFresponsive nociceptors. While the localization pattern of the B 1 receptor was not clear in the TG cryosections, weak immunoreactivity for B 1 receptors was observed in the primary cultured TG neurons. The application of BK activated B 2 receptors and Lys-[Des-Arg 9 ]BK activated the B 1 receptors. B 2 receptor activation mobilized [Ca 2+ ] i by releasing Ca 2+ from internal Ca 2+ stores with partial Ca 2+ influx from the extracellular medium. B 2 receptors, which are expressed ubiquitously and constitutively in healthy tissues, are essential in the early stages of general pain generation (Hall, 1992). The constitutive expression of B 2 receptors in TG neurons has been studied by reverse transcription-polymerase chain reaction (RT-PCR) analyses (Ceruti et al., 2011) and immunocytochemical analyses in cultured TG neurons (Patwardhan et al., 2005). Although BK-induced [Ca 2+ ] i increases have also been reported in TG neurons (Ceruti et al., 2008(Ceruti et al., , 2011, precise functional expression patterns of B 1 and B 2 receptors in TG neurons remained unclear. The results of the present study showing the functional expression and localization of B 2 receptors in TG neurons were in line with the previous results. The results of this study were also in line with the pharmacological properties of BK, which is a potent and endogenous agonist for B 2 receptors and not B 1 receptors in the sympathetic neurons of the rat superior cervical ganglion (Babbedge et al., 1995) and in Chinese hamster ovary (CHO) cells stably expressing recombinant human B 1 or B 2 receptors (Simpson et al., 2000). Furthermore, BK has an affinity for B 2 receptors that is 500 times that for B 1 receptors (Simpson et al., 2000). Therefore, B 2 receptors are histologically and functionally expressed, and endogenous BK preferentially activates B 2 receptors in rat TG neurons.
The expression of the B 1 receptor, which is induced as a result of tissue damage and inflammation, is involved in chronic inflammation or tissue injury (Hall, 1992). The observations of the constitutive B 1 receptor expression in TG and dorsal root ganglion (DRG) neurons have been inconsistent. In DRG neurons, some immunohistochemical studies have reported constitutive B 1 receptor expression (Ma et al., 2000;Wotherspoon and Winter, 2000). In contrast, other studies have described that B 1 receptor activation-induced [Ca 2+ ] i responses could not be observed in DRG neurons (Brand et al., 2001). In TG neurons, an immunohistochemical study has shown the constitutive expression of B 1 receptors (Ma et al., 2000). In contrast, RT-PCR analyses have demonstrated that B 1 receptor mRNA was barely expressed in intact tissue, while it was weakly  expressed in primary cultured TG neurons. In primary cultured TG neurons, the levels of expression of B 1 receptor mRNA have been reported to depend on the length of the culture period (Ceruti et al., 2011). The present immunohistochemical and immunocytochemical results were similar to the previous RT-PCR results; B 1 receptor immunoreactivity was weakly positive in cultured TG neurons and could not be detected in intact TG tissue. Although few report concerning B 1 receptor-induced [Ca 2+ ] i response in TG neurons exist, in the [Ca 2+ ] i imaging in the present study, the B 1 receptor agonist, Lys-[Des-Arg 9 ]BK which is a metabolite of endogenous BK in peripheral tissues (Regoli et al., 2001), dose-dependently increased [Ca 2+ ] i in the presence of extracellular Ca 2+ , and this increase was suppressed by a B 1 receptor-specific antagonist (Figures 5C-F). These results of B 1 receptor expression in primary cultured TG neurons suggest that the expression of B 1 receptors is induced in TG neurons by tissue damage and/or inflammation. However, further studies are required to evaluate the expression patterns of B 1 receptors in native TG neurons.
BK-induced [Ca 2+ ] i increases were observed in both the presence and absence of extracellular Ca 2+ . However, the amplitudes of the [Ca 2+ ] i increases in the absence of extracellular Ca 2+ were significantly smaller (84.9 ± 11.3%, N = 161) than those in the presence of Ca 2+ (100%; Figures 4A,D). This indicated that the BK-induced [Ca 2+ ] i mobilization (by B 2 receptor activation) was mainly composed of Ca 2+ release from internal stores with partial Ca 2+ influx from the extracellular medium. Notably, BK has been reported to activate voltage-dependent Ca 2+ channels in rat submucosal plexus neurons (Avemary and Diener, 2010;Rehn et al., 2013), and transient receptor potential cation channel subfamily-V member-1 channels in rat DRG neurons (Ferreira et al., 2004;Mistry et al., 2014). However, BK-induced Ca 2+ currents could not be recorded in TG neurons (Kitakoga and Kuba, 1993). Although further studies are needed to clarify which Ca 2+ influx pathways contribute to the BK-induced Ca 2+ influx in TG neurons, the present results clearly indicate that BK mobilizes [Ca 2+ ] i through both intracellular Ca 2+ release and Ca 2+ influx.
In addition, NGF-TrkA signaling plays important roles in not only the developmental processes of peptidergic nociceptive afferents, but also in the generation of acute and chronic pain state in adults. The signaling also upregulates B 2 receptor expression in peptidergic nociceptors (Mantyh et al., 2011). Thus, the results showing colocalization of B 2 receptor and TrkA immunoreactivity in TG neurons strongly support reports describing that the B 2 receptor mediates inflammatory/neuropathic pain induced by peripheral sensitization in the orofacial region (Chichorro et al., 2004;Luiz et al., 2010); however, the present results obtained from neonatal rat may not reflect the situation in adults.
In conclusion, B 2 receptors were expressed constitutively, and their activation induced the mobilization of [Ca 2+ ] i by releasing Ca 2+ from intracellular stores with partial Ca 2+ influx. In contrast, B 1 receptor expression was faint in cultured TG neurons and absent in neurons in TG cryosections, although a metabolite of endogenous BK elicited [Ca 2+ ] i increases. These results indicated that both BK and its metabolites activated [Ca 2+ ] i mobilization in TG neurons through B 2 and B 1 receptor activation, respectively.