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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Action of Bradykinin in the Submucosal Plexus of Guinea Pig Small Intestine

Department of Physiology and Cell Biology, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio

Received August 27, 2003; accepted December 18, 2003.

	Abstract

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Intracellular recording methods with "sharp" microelectrodes were used to study actions of bradykinin (BK) on electrical behavior of morphologically identified neurons and the identification and localization of BK receptors in the submucosal plexus of guinea pig small intestine. Exposure to BK depolarized the membrane potential and elevated excitability in submucosal neurons with AH-type electrophysiological behavior and Dogiel II multipolar morphology and in neurons with S-type electrophysiological behavior and uniaxonal morphology. BK-evoked depolarizing responses were associated with increased neuronal input resistance in AH-type neurons and decreased input resistance in S-type neurons. The selective B₂ BK receptor antagonists HOE-140 (icatabant acetate) and WIN64338[(S)-4[2-bis(cyclohexylamino)methyleneamino]-3-(2-napthalenyl)-1-oxopropylamino]benzyl tributyl phosphonium chloride hydrochloride], but not the selective B₁ receptor antagonists des-arg¹⁰-HOE-140 and des-arg⁹-leu⁸-BK, suppressed the BK-evoked responses. The selective B₂ receptor agonist Kallidin, but not the selective B₁ receptor agonist des-arg⁹-BK mimicked the excitatory action of BK. Western blot analysis and reverse transcription-polymerase chain reaction confirmed the expression of B₂ receptor protein and mRNA. Binding studies with a fluorescently labeled BK₂ antagonist found expression of B₂ receptors on a majority of the ganglion cells. B₂ receptors occupied 82% of the neurons that expressed immunoreactivity for neuropeptide Y, 75% of the neurons that expressed vasoactive intestinal peptide, 84% of the neurons that expressed substance P, 71% of the neurons that expressed choline acetyltransferase, and all neurons that expressed calbindin immunoreactivity. The results suggest that the B₂ receptor mediates the excitatory action of BK on submucosal plexus neurons. Pathophysiological significance of the excitatory actions on secretomotor neurons might be stimulated mucosal secretion and the secretory diarrhea associated with intestinal inflammatory states.

The present study was focused on bradykinin (BK) as one of the putative mediators formed during inflammatory, ischemic, and hemorrhagic states in the bowel (Zeitlin and Smith, 1973; Brown and Roberts, 2001). It was the second phase of a project to understand how BK formation might alter gut motility and secretion through actions in the ENS. The second phase aimed to study the cellular neuropharmacology of BK on neuronal elements of the submucosal plexus with a view toward potential pathological influence on intestinal secretory function. The earlier phase investigated actions, mechanisms of action, and localization of action to specific categories of neurons in the myenteric division of the ENS (Hu et al., 2003). Acquisition of information for each of the two plexuses individually was advised because the functional neurobiology differs for each plexus. Examples of differences include the segregation of motor neurons and specific reflex behaviors between the two plexuses. Excitatory and inhibitory musculomotor neurons are located in the myenteric plexus, and secretomotor neurons are in the submucosal plexus. Reflex circuits for propulsive motility are "hardwired" into the myenteric plexus (Smith et al., 1990; Kunze and Furness, 1999). Reflex circuitry for the control of intestinal secretion is incorporated in the neural networks of the submucosal plexus (Frieling et al., 1992; Cooke, 2000). Earlier studies in the myenteric plexus found excitatory actions of BK on neurons with AH-type 2 electrophysiological behavior, Dogiel type II multipolar morphology, and immunoreactivity for calbindin and on neurons with S-type electrophysiological behavior, uniaxonal Dogiel type I morphology and immunoreactivity for nitric-oxide synthase (Hu et al., 2003). Calbindin-positive Dogiel type II neurons predominate in the myenteric plexus and are scarce in the submucosal plexus (Brookes 2001a,b). Nitric oxide-positive uniaxonal neurons that express nitric-oxide synthase and project their axon in the aboral direction within the myenteric plexus are identified as inhibitory motor neurons to the musculature (Brookes, 2001a,b). Stimulation of excitability in this neuronal population suppresses contractile activity. A subpopulation of uniaxonal neurons that express immunoreactivity for choline acetyltransferase, neuropeptide Y, and vasoactive intestinal peptide in the submucosal plexus are identified as secretomotor neurons (Bornstein and Furness, 1988). Elevated activity in this neuronal population increases mucosal secretion of H₂O and electrolytes, which when exaggerated, leads to neurogenic secretory diarrhea (Cooke, 1994). One of the aims of the present study was to evaluate actions of BK on secretomotor neurons in the submucosal plexus.

Bradykinin B₁ and B₂ receptors mediate the actions of BK. Both receptors have been defined pharmacologically by use of specific peptide and nonpeptide antagonists. DNA that encodes functional BK B₂ receptors from human, rat, rabbit, mouse, and guinea pig sources have been cloned (McEachern et al., 1991; Hess et al., 1992; Ma et al., 1994; Bachvarov et al., 1996; Farmer et al., 1998). Bradykinin binds preferentially to the B₂ BK receptor and the related kinins generated by cleaving the terminal arginine residue from kallidin and BK (e.g., des-arg¹⁰-kallidin, and des-arg⁹-bradykinin) bind preferentially to the BK B₁ receptor. An aim of the present study was to identify the kind of BK receptor expressed by neurons in the submucosal plexus.

	Materials and Methods

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Adult male Hartley-strain guinea pigs (300-350 g) were stunned by a sharp blow to the head and exsanguinated from the cervical vessels according to protocols approved by The Ohio State University Laboratory Animal Care and Use Committee and United States Department of Agriculture Veterinary Inspectors. Submucosal plexus preparations were obtained from segments of small intestine removed 10 to 20 cm proximal to the ileocecal junction. The segments were microdissected for electrophysiological recording and fluorescence immunohistochemistry as described previously (Frieling et al., 1991).

Electrophysiology. The preparations were placed in 2-ml chambers and superfused with Krebs' solution warmed to 37°C and gassed with 95% O₂,5%CO₂ (pH 7.3-7.4), at a rate of 10 to 15 ml min^-1. The composition of the Krebs' solution was 120 mM NaCl, 6 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, 1.35 mM NaH₂PO₄, 14.4 mM NaHCO₃, and 11.5 mM glucose. Transmembrane electrical potentials were recorded with conventional "sharp" intracellular microelectrodes filled with 4% biocytin (Sigma-Aldrich, St. Louis, MO) in 2 M KCl or potassium acetate containing 0.05 M Tris buffer (pH 7.4) and having resistances of 80 to 200 M. Methods for electrophysiological recording of electrical behavior in submucosal neurons and procedures for development of the intraneuronal marker biocytin were the same as described previously (Liu et al., 2003a).

Reverse Transcriptase-Polymerase Chain Reaction. Total RNA was extracted from submucosal plexus preparations with TRIzol (Invitrogen, Carlsbad, CA). Reverse transcription (1 µg of RNA) was performed by adding the following and incubating at 42°C for 60 min: 2.0 µl of 10x RT buffer (0.1 M Tris, 0.5 M KCl, pH 8.3), 4.0 µl of 25 mM MgCl_2, 2.0 µl of deoxynucleotide mix (dATP, dCTP, dGTP, and dTTP, each at 10 mM), 2.0 µl of oligo-p(dT) primer (0.8 µg/µl), 50 units of RNase inhibitor, 20 units of avian myeloblastosis virus reverse transcriptase (Roche Diagnostics, Indianapolis, IN), and 1 µl of total RNA. Heating to 99°C for 5 min and then cooling to 4°C for 5 min deactivated the enzyme. The following reaction mixture was added to 5 µl of the cDNA product: 25 µl of PCR master mix (Roche Diagnostics), 2.0 µl of primers, and 18 µl of PCR grade sterile water. The primers used were as follows.

1. Upstream: 5'-CGTTCTTGTGGGTCCTGTTT-3'
   
2. Downstream 5'-CAGGAGGACATTGGTGAACA-3'
   
3. Upstream: 5'-TATTCCTGGTGGTGGCTATC-3'
   
4. Downstream: 5'-GGAGGCTACCAGTGTGAGGA-3'
   

Primers 1 and 2 were designed from the mRNA of a guinea pig BK B₂ receptor (GenBank accession no. AJ003243 [GenBank] ) to amplify a 500-base pair fragment. Primers 3 and 4 were a pair of degenerate primers used to amplify a 401-base pair fragment for the guinea pig BK B₁ receptor. Primers 3 and 4 were made from the conserved region between mouse BK B₁ receptor (GenBank accession no. NM_007539 [GenBank] ) and rat BK B₁ receptor (GenBank accession no. U66107 [GenBank] ). Amplification was done with an iCycler (Bio-Rad, Hercules, CA). PCR cycles consisted of denaturation for 1 min at 94°C, annealing at 56°C for 90 s, and extension at 72°C for 90 s. A total of 30 cycles was followed by completion of extension for 7 min at 72°C. PCR product (12 µl) was analyzed by electrophoresis on 2% agarose gel. Sequencing was done with an ABI 373XL DNA sequencer after the PCR product was purified with Qiaquick PCR purification kit (QIAGEN, Valencia, CA). RT-PCR without avian myeloblastosis virus reverse transcriptase was used as a negative control.

Western Blot. Submucosal plexus preparations were prepared as described above, washed in cold Krebs' solution, and stored at -70°C. The Complete Mini kit (Roche Diagnostics) was used to obtain samples of total submucosal plexus protein. Samples of total protein (20-100 µg) were loaded onto 11% sodium dodecyl sulfate polyacrylamide gels and blotted onto nitrocellulose filters. The filters were incubated with antiserum in buffer containing 5% nonfat dairy milk for 4 h at room temperature. A rabbit polyclonal antibody specific for the B₁ bradykinin receptor (1:100, code SC-15048; Santa Cruz Biotechnology Inc., Santa Cruz, CA) or a monoclonal B₂ receptor antibody (1:1000 code: 610451; BD Transduction Laboratories, San Diego, CA) was used. The filters were then incubated with goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Antibody labeling was detected by chemiluminescence. Color molecular weight standards were run on each gel.

Immunohistochemistry. Whole-mounts of the submucosal plexus were incubated in 10% normal horse serum in PBS for 1 h at room temperature before exposure to the primary antisera diluted in hypertonic PBS containing 10% normal horse serum, 0.3% Triton X-100, and 0.1% sodium azide. The preparations were placed in humidified chambers and processed for indirect single or double immunofluorescence staining by incubation for 18 h at room temperature in rabbit or mouse antiserum or a mixture of primary antibodies from different species to achieve double labeling. The primary antibodies used were mouse anti-B₂ receptor (1:1000) (code 610451; BD Transduction Laboratories), rabbit anti-B₂ receptor (1:1000) (code AS276; kindly provided by Prof. Müller-Esterl, Frankfurt, Germany), mouse anti-HuC/HuD neuronal protein (1:200) (code A21271 [GenBank] ; Molecular Probes, Eugene, OR), goat anti-choline acetyltransferase (ChAT) (1:100) (code AB 144P; Chemicon International, Temecula, CA); sheep anti-neuropeptide Y (NPY) (1: 3000) (code AB1583; Chemicon International), rat anti-substance P (SP) (1:200) (code MAB356; Chemicon International), rabbit anti-vasoactive intestinal peptide (VIP) (1:100) (code IHC7161; Peninsula Laboratories, Belmont, CA), and goat anti-calretinin (1:5000) (code AB1550; Chemicon International). The preparations were incubated for 24 h in the primary antibody at room temperature followed by 3 x 1-min washes with PBS and then incubated in fluorescein isothiocyanatelabeled donkey anti-rabbit secondary IgG (code 711-095-152), donkey anti-sheep secondary IgG (code 713-095-147), donkey anti-rat secondary IgG (code AP189F) or donkey anti-goat secondary IgG (code 705-095-147), and Cy3-labeled donkey anti-rabbit secondary IgG (code 711-165-152) or donkey anti-mouse secondary IgG (code 715-165-150) for another 30 min at 37°C followed by 3 x 10-min washes with PBS. All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The preparations were cover-slipped in Vectorshield (Vector Laboratories, Burlingame, CA). Fluorescent labeling was examined with a Nikon Eclipse E-600 fluorescent microscope that was equipped with appropriate filters and a SPOT-2 chilled color and B/W digital camera (Diagnostic Instruments, Sterling Heights, MI).

Drugs and Chemicals. Pharmacological agents were applied either by addition to the bathing solution or by micropressure ejection from micropipettes (10-20-µm tip diameter) positioned within 200 µm of the impaled neuron. Tetrodotoxin, N-methyl-D-glucamine, choline chloride, BK, des-arg⁹-BK, des-arg⁹-leu⁸-BK, indomethacin, kallidin, and piroxicam were obtained from Sigma-Aldrich. HOE-140 and des-arg¹⁰-HOE-140 were purchased from Sigma/RBI (Natick, MA). WIN64338was purchased from Tocris Cookson Inc. (Ballwin, MO). HOE-741 was obtained from PerkinElmer Life Sciences (Boston, MA). For experiments involving low Na⁺ Krebs' solution, Na⁺ was substituted with equal molar N-methyl-D-glucamine or choline chloride. The substitution reduced the Na⁺ concentration of the bathing solution from 146.2 to 26.2 mM.

Data Analysis. Data are presented as means ± S.E.M. Concentration-response curves were constructed using the following least-squares fitting routine. V = V_max/[1 + (EC₅₀/C)^nH], where V is the observed membrane depolarization, V_max is the maximal response, C is the corresponding drug concentration, EC₅₀ is the concentration that evoked a half-maximal response, and n_H is the apparent Hill coefficient. Antagonist concentration-inhibition curves were obtained in individual cells by using a fixed concentration of BK (30 nM), and progressively increasing the concentration of antagonist. IC₅₀ values were calculated by least-squares fitting to the following equation. V = V_max/[1 + (IC₅₀/[Ant])^-nH], where V is the observed percentage of inhibition and V_max is peak percentage of inhibition, [Ant] is the corresponding antagonist's concentration, IC₅₀ is the antagonist concentration that produced half-maximal inhibition of BK-evoked responses, and n_H is the apparent Hill coefficient. The graphs in the figures were drawn by averaging results from all experiments and fitting a single concentration-response curve to the pooled data with SigmaPlot software (SPSS Science Inc., Chicago, IL). All EC₅₀ and IC₅₀ values are the mean ± S.E.M.

	Results

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Identification of the Submucosal B₂ Receptor. An RT-PCR product with 500 nucleotide base pairs was amplified by a pair of specific primers for the guinea pig BK B₂ receptor (Fig. 1A, lane 2). No RT-PCR product was amplified for the B₁ receptor (Fig. 1A, lane 3). The nucleotide sequence for the RT-PCR product was purified and sequenced. The sequence aligned with that of guinea pig lung BK B₂ receptor cDNA. There was complete identity with the corresponding region of the guinea pig lung B₂ receptor (from 245 to 744) (GenBank accession no. AJ 003243).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Bradykinin B2 receptor expression in guinea pig small intestinal submucosal plexus. A, RT-PCR analysis with primers specific for the B2 receptor identified the appropriate 500-base pair band. A pair of specific primers for the B1 receptor did not detect expression of the bradykinin B1 receptor. Lane 1 is a DNA calibration ladder. Lane 2 shows the RT-PCR product obtained with specific primers for the B2 receptor. Absence of expression with specific primers for the B1 receptor is in lane 3. B, Western blot analysis with a polyclonal antibody to the bradykinin B1 receptor found no evidence for expression of the B1 receptor (lane 1). Analysis with a monoclonal bradykinin B2 receptor antibody generated a 42-kDa band. Lane 3 is a positive B2 receptor control obtained for rat pituitary gland. The known difference in molecular mass of 3-kDa between the rat and guinea pig BK B2 receptor accounts for differences between lanes 2 and 3.

Western blot analysis was used to identify the presence of B₂ receptor protein in submucosal protein extracts. A mouse anti-B₂ receptor antibody recognized a protein band of 42 kDa in guinea pig submucosal protein extracts (Fig. 1B, lane 2). The positive control (Fig. 1B, lane 3) was obtained from a rat pituitary protein extract. The difference in molecular mass of 3 kDa between the rat and guinea pig BK B₂ receptor accounts for differences seen on Western blots (SWISSPROT; O70526 [GenBank] for guinea pig and P25023 [GenBank] for rat). Expression of B₁ receptor protein was not detected in submucosal plexus extracts (Fig. 1B, lane 1).

BODIPY 576/579 is a commercially available fluorescent probe that can be bound to a variety of compounds, including the selective BK B₂ receptor antagonist HOE-140. HOE-741 is the BODIPY 576/579 tagged form of HOE-140. We used 30 nM HOE-741 as a tool for localizing BK B₂ receptors in submucosal ganglia (Fig. 2A). Pretreatment with 300 nM HOE-140 for 40 min before application of HOE-741 for 40 min quenched fluorescent labeling by HOE-741 and enhanced confidence in HOE-741 selectivity for labeling of cells that expressed BK B₂ receptors (Fig. 2B). Fluorescent labeling with HOE 741 was localized exclusively to ganglion cell somas and a limited number of neuronal processes (Fig. 2A). The specific neuronal localization was confirmed by double labeling with anti-HuC/HuD neuronal protein mouse monoclonal antibody that has been shown to label all enteric neurons (Hu et al., 2002; Lin et al., 2002) (Fig. 2C).

View larger version (36K): [in this window] [in a new window]   Fig. 2. Localization of the bradykinin B2 receptor in the guinea pig small intestinal submucosal plexus. A, localization of HOE-741 binding to neuronal cell somas and proximal processes. B, preincubation with the selective B2 antagonist HOE-140 before exposure to HOE-741 blocked binding of HOE-741 to the B2 receptor. C, double labeling of HOE-741 with anti-HuC/HuD, which labels all neurons, suggests that the bradykinin B2 receptor was expressed exclusively by submucosal ganglion cells. D to F, immunoreactivity for NPY was colocalized with HOE-741 fluorescence. G and I, immunoreactivity for VIP colocalized with HOE-741 fluorescence.

The chemical codes for submucosal neurons with BK B₂ receptors were studied by double labeling HOE-741 with immunoreactivity for ChAT, NPY, VIP, calretinin, calbindin, or SP. HOE-741 fluorescence was found in 82.4% (1103/1339) of the ganglion cells associated with NPY immunoreactivity (Fig. 2, D-F).

Most of the neurons (74.6%; 1894/2540) with immunoreactivity for VIP also expressed the B₂ receptor (Fig. 2, G-I). A large number of SP-immunoreactive neurons (83.6%; 424/507) and all calbindin-immunoreactive neurons were labeled by HOE-741 and 70.5% (2403/3409) of the ChAT-immunoreactive neurons were labeled with HOE-741. Colocalization of HOE-741 binding with calretinin immunoreactivity was not found (data not shown).

Indirect immunofluorescence with a mouse anti-B₂ receptor antibody (code 610451; BD Transduction Laboratories) was used to study the distribution pattern of the BK B₂ receptor and as a control for the results with HOE-741. The distribution pattern for the B₂ receptor with the anti-B₂ receptor antibody was similar to that found with HOE-741 (Fig. 3). All NPY neurons (n = 2135) expressed BK B₂ receptor immunoreactivity (Fig. 3, A-C). NPY-immunoreactive neurons are known to be a subset of cholinergic neurons in the guinea pig submucosal plexus (Brookes, 2001a,b). Double labeling of BK B₂ receptor immunoreactivity with immunoreactivity for ChAT and VIP showed localization of the B₂ receptor with both ChAT-(data not shown) and VIP-positive neurons (Fig. 3, D-F). Ninety-nine percent of neurons in guinea pig submucosal ganglia express ChAT or VIP (Brookes, 2001a,b). Our finding that the BK B₂ receptor is colocalized with both ChAT and VIP might suggest that 99% of the neuronal population in the submucosal plexus express the B₂ receptor.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Bradykinin B2 receptor immunoreactivity in the guinea pig small intestinal submucosal plexus. A to C, bradykinin B2 receptor immunoreactivity was colocalized with NPY. D to F, double labeling of B2 receptor immunoreactivity with VIP. All VIP immunoreactive neurons expressed B2 receptor immunoreactivity.

As a separate control, we also used a rabbit polyclonal B₂ receptor antibody (code AS276) that was characterized by Reyes-Cruz et al. (2000). The same immunostaining pattern for distribution of the B₂ receptor was obtained with this antibody as with the mouse antibody described above (data not shown).

Electrophysiology. Submucosal neurons were classified as AH- or S-type based on generally accepted criteria (Bornstein et al., 1994; Wood, 1994a). Application of BK (0.3-300 nM) by addition to the superfusion solution for 50 s evoked a concentration-dependent depolarization of the membrane potential coincident with enhanced excitability in 23 of 26 AH-type neurons and 59 of 71 S-type neurons (Fig. 4A). Depolarizing responses in AH-type neurons were associated with an increase in neuronal input resistance and with decreased input resistance in S-type neurons. The electrophysiological results were consistent with the histological finding that most submucosal ganglion cells express the BK receptor. The depolarizing responses began after a 5- to 10-s delay and peaked in 2.5 to 3 min. Enhanced excitability was reflected by an increased number of action potentials evoked by intraneuronal injection of constant current depolarizing pulses, anodal break excitation at the offset of hyperpolarizing current pulses, and the occurrence of spontaneous spike discharge. Suppression of the characteristic hyperpolarizing after-potentials in AH-type neurons was another action of BK (data not shown). The effects of BK were fully reversible after a 10- to 20-min washout period. Time for recovery during washout was directly related to the concentration. No apparent desensitization was observed with repeated exposures at 10- to 20-min intervals. Bradykinin-evoked responses persisted when 300 nM tetrodotoxin was coapplied with BK in seven S-type neurons. Likewise, reduction of Ca²⁺ to 0.5 mM and elevation of Mg²⁺ to 12.5 mM in the bathing solution did not suppress the amplitude of BK-evoked depolarizing responses. Tetrodotoxin blocks generation of action potentials in ENS axons and the axonal release of neurotransmitters (Wood, 1994b). Elevated Mg²⁺ and reduced Ca²⁺ prevent Ca²⁺ entry into axon terminals and thereby suppress the release of neurotransmitters. Failure of tetrodotoxin or reduced Ca²⁺ and elevated Mg²⁺ to alter the excitatory responses to BK was indicative of a direct action at receptors on the ganglion cell rather than an indirect action due to excitation of neighboring neurons that provided synaptic input to the recorded ganglion cell.

View larger version (45K): [in this window] [in a new window]   Fig. 4. Depolarizing responses to bradykinin were concentration-dependent in guinea pig small intestinal submucosal neurons. A, responses to BK in concentrations from 0.3 to 300 nM in the same neuron. Downward deflections are electrotonic potentials evoked by intraneuronal injection of constant current hyperpolarizing pulses. Decreased amplitude of the downward deflections reflects decreased input resistance. Upward deflections are action potentials occurring at the offset of hyperpolarizing current pulses. Occurrence of action potentials reflects elevated neuronal excitability. Action potential amplitude is blunted by the low-frequency response of the chart recorder. B, concentration-response relationship for BK-evoked depolarizing responses. Data were obtained from seven S-type neurons. C, morphology of the uniaxonal neuron from which the records in A were obtained.

Concentration-Response Relations (S-Type Neurons). Concentration-response relations for BK-evoked depolarizing responses were determined only for S-type neurons due to scarcity of AH-type neurons in the guinea pig submucosal plexus.

Threshold concentration for a measurable effect of BK on the membrane potential was 0.1 to 3 nM. The EC₅₀ was 2.7 ± 0.4 nM for seven S-type neurons and the maximal depolarizing response was 18.2 ± 2.5 mV (n = 9 S-type neurons) evoked by 300 nM BK (Fig. 4, A and C). The actions of the selective BK B₁ receptor antagonists des-arg⁹-leu⁸-BK and des-arg¹⁰-HOE-140 were compared with the actions of the selective BK B₂ receptor antagonists HOE-140 and a nonpeptide B₂ receptor antagonist, WIN64338 HOE-140 (1-300 nM) concentration dependently suppressed the depolarizing action of 30 nM BK with an IC₅₀ value of 31.0 ± 6.0 nM for eight S-type neurons (Fig. 5, A-A₇). The threshold concentration for HOE-140 was 1 to 3 nM. The action of HOE-140 was long-lasting, requiring up to 40 min of washout for full recovery. WIN64338(0.1-3 µM) also suppressed BK-evoked depolarization in concentration-dependent manner with an IC₅₀ value of 0.7 ± 0.1 µM in seven S-type neurons. The IC₅₀ value for WIN64338was 20-fold higher than for HOE-140 (Fig. 5E). Neither 300-nM des-arg⁹-leu⁸-BK in five ganglion cells nor 300 nM des-arg¹⁰-HOE-140 in seven ganglion cells suppressed BK-evoked responses. The selective BK B₂ receptor agonist kallidin (10 nM-3 µM) acted to depolarize the membrane potential with an EC₅₀ value of 298.9 ± 61.5 nM for five S-type neurons. On the other hand, the selective B₁ receptor agonist des-Arg⁹-BK was ineffective in concentrations of 1 to 3 µM in nine neurons (Fig. 5C).

View larger version (54K): [in this window] [in a new window]   Fig. 5. Bradykinin-evoked responses were mediated by the BK B2 receptor. A1 to A7, HOE-140, a selective B2 receptor antagonist, suppressed responses to 30 nM BK in a concentration-dependent manner. Washout reversed action of 300 nM HOE-140. B, WIN64338 a selective nonpeptide bradykinin B2 receptor antagonist, abolished responses to 30 nM BK. C, Des-arg9-BK, a selective bradykinin B1 receptor agonist, did not mimic the action of bradykinin. D, Des-arg10HOE-140, a selective bradykinin B1 receptor antagonist, did not suppress the action of bradykinin. Upward deflections are action potentials occurring at the offset of hyperpolarizing current pulses. Occurrence of action potentials reflects elevated neuronal excitability. Neuron from which the records in A to D were obtained is in the inset. F, concentration-response relations for suppression of responses to 30 nM bradykinin by HOE-140 and WIN64338 Data for HOE-140 were from eight S-type neurons, and data for WIN64338were obtained from seven S-type neurons. The IC50 value for HOE-140 was 31.0 ± 6.0 nM and the IC50 value for WIN64338was 0.7 ± 0.1 µM.

Ionic Mechanisms. Depolarizing responses evoked by BK (0.3-300 nM) were associated with increased neuronal input resistance in 22 of 75 neurons and with decreased input resistance in 39 of 75 neurons. Input resistance was unchanged in 14 of 75 neurons. The input resistance was decreased or unchanged in S-type neurons with uniaxonal morphology and increased in AH-type neurons with multipolar morphology.

Figure 6A shows the current-voltage (I-V) relationship for an S-type neuron in which the depolarizing responses to BK were associated with decreased input resistance. The I-V curves obtained in the absence and presence of BK intersected at 8 mV, which is the estimated reversal potential for the depolarizing response. The mean extrapolated reversal potential for the BK-evoked depolarizing responses associated with decreased input resistance was -10.8 ± 8.4 mV (range -28 to +10 mV) for five S-type neurons.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Reversal potentials for bradykinin-evoked depolarizing responses differed for S- and AH-type submucosal neurons. A, current-voltage relationship for an S-type neuron in the presence and absence of 30 nM bradykinin. Reduced slope of the curve for bradykinin reflects decreased input resistance. Intersection of the two curves at a membrane potential of 10 mV reflects the reversal potential. B, current-voltage relationship for an AH-type neuron in the presence and absence of 30 nM BK. Increased slope of the curve for bradykinin reflects increased input resistance. Intersection of the two curves at a membrane potential of approximately -92 mV reflects the reversal potential. C, micropressure application of 100 nM bradykinin (puffs) evoked slowly activating membrane depolarization in an S-type neuron. The amplitude of the depolarizing responses progressively decreased as the resting membrane potential was current-clamped in the depolarizing direction starting at -83 mV and continuing in steps that stopped at 23 mV. D, data obtained from four S-type neurons in which the depolarizing responses to bradykinin were associated with decreased input resistance. The data extrapolate to a reversal potential between -20 and 20 mV (mean = 6.5 ± 8.6 mV).

Application of BK by pressure microejection ("puffs") evoked slowly activating and prolonged depolarizing responses that were similar to responses evoked by bath application (n = 10 neurons). No rapidly activating depolarizing responses similar to nicotinic responses to acetylcholine were ever observed. The prolonged depolarization in response to puffs of BK was associated with the firing of action potentials (n = 7 neurons). The input resistance decreased in most of the neurons tested (6/10) with pressure microejection of 100 nM BK. The amplitude of the BK-evoked depolarizing responses that were associated with decreased input resistance increased when the membrane potential was current-clamped in the hyperpolarizing direction and decreased when the membrane potential was shifted in the depolarizing direction (Fig. 6, C and D).

Electrophysiological behavior of this nature suggested that BK activates a cationic conductance in the S-type neurons. We tested this hypothesis by examining BK-evoked depolarizing responses in bathing solutions in which the concentration of Na⁺ was reduced by 82% by substitution of NaCl with N-methyl-D-glucamine or choline chloride. The membrane potential was current-clamped at its original level before application of BK in the studies with reduced Na⁺. In low Na⁺ solution, the amplitude of depolarizing responses evoked by 100 nM BK were reduced by 50.7 ± 5.8% in seven S-type neurons. BK evoked no spike discharge in low Na⁺ solution (data not shown).

Suppression of Ca²⁺ entry in bathing solutions containing 16 mM Mg²⁺ and with Ca²⁺ reduced from 2.5 mM to 1 mM did not significantly reduce the amplitude of the depolarizing responses to BK in S-type neurons. This suggested that the depolarizing responses to BK in S-type neurons might not be dependent on influx of extracellular Ca²⁺.

Exposure to BK evoked membrane depolarization associated with increased input resistance in 22 AH-type neurons. An I-V relationship for an AH-type neuron in the presence and absence of BK is shown in Fig. 6B.

I-V curves obtained in the absence and presence of 30 nM BK intersected at -88 mV. The mean reversal potential for the depolarizing responses that were associated with increased input resistance was -92.0 ± 4.5 mV (range -85 to -105 mV) for five AH-type neurons. These values are near the estimated equilibrium potential for K⁺ in enteric neurons and may reflect decreased K⁺ conductance during the depolarizing responses to BK in AH-type neurons (North, 1973).

	Discussion

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Bradykinin is formed in inflammatory, ischemic and hemorrhagic conditions in the gut (Brown and Roberts, 2001). It is a peptide cleaved from ₂ globulins termed kininogens. Specific proteases called kallikreins release bradykinin from the kininogens (Bhoola et al., 1992). The present studies tested the suggestion that after intramural formation in the intestine, BK might act on the ENS as a paracrine mediator to alter neural control of secretory and motility functions at the organ level.

Excitation of ganglion cells in the submucosal plexus was found to be a primary action of bradykinin. Several lines of evidence suggested that the BK B₂ receptor was the exclusive mediator of the neuronal excitatory responses. No evidence for involvement of the BK B₁ receptor was obtained. The evidence for BK B₂ involvement included blockade of BK-evoked responses by the selective BK B₂ receptor antagonist HOE-140, lack of effect of selective BK B₁ receptor antagonists, selective binding of fluorescently labeled HOE-741 to submucosal ganglion cells, and expression of the mRNA transcript and receptor protein for the B₂, but not the B₁ receptor in the submucosal plexus. The evidence is consistent with earlier published reports that the B₂ receptor is the sole receptor for BK in the guinea pig (Kachur et al., 1987; Pruneau et al., 1995). A similar conclusion was reached for the mouse where knockout of the B₂ receptor eliminated all responses to BK in both in vivo and in vitro preparations (Borkowski et al., 1995; Seabrook et al., 1997).

The B₂ receptor, unlike the B₁ receptor, is constitutively expressed in most tissues; B₁ receptors are not constitutively expressed. Bradykinin B₁ receptors are up-regulated by inflammation and cytokines (Dray and Perkins, 1993). This leaves open the untested possibility that enteric neurons might be found to express the B₁ receptor if examined in intestinal inflammatory states.

Results from both electrophysiological and histological studies suggested that the majority of neuronal cell bodies in the ganglia expressed the B₂ receptor. Bradykinin selectively stimulated excitability in neurons that expressed immunoreactivity for VIP and/or ChAT. VIPergic and cholinergic neurons with S-type electrophysiological behavior and uniaxonal morphology in the submucosal plexus are known to function as secretomotor neurons (Brookes, 2001a,b). Enhanced excitability in the population of secretomotor neurons elevates intestinal mucosal secretion of electrolytes, H₂O, and mucus and can underlie the neurogenic secretory diarrhea often associated with inflammatory states and ischemia-reperfusion injury (Cooke, 2000).

Exposure to BK evoked depolarizing responses in submucosal neurons with S-type electrophysiological behavior and uniaxonal morphology, and the depolarization was associated with decreased neuronal input resistance. A reversal potential near -10 mV for the depolarizing responses suggested that the underlying ionic mechanism for the responses in the S-type neurons might be opening of nonselective cationic conductance channels. Findings that the amplitude of the BK-evoked responses was reduced in bathing solution with reduced Na⁺ and remained unaffected in solutions with reduced Ca²⁺ and elevated Mg²⁺ were consistent with elevated Na⁺ (and perhaps K⁺) conductance being the ionic mechanism for the depolarizing responses. The responses to BK in S-type neurons were similar to reported actions for substance P, serotonin, and acetylcholine in the submucosal plexus, which were interpreted by Shen and Surprenant (1993) to be due to an increase in a cation-selective ("mainly sodium") conductance.

AH-type neurons were not a primary focus for the present study because their numbers in the submucosal plexus were less than 10% of the neuronal population. Nevertheless, both direct and indirect immunofluorescence studies confirmed the expression of BK B₂ receptors by submucosal neurons with a combination of AH-type electrophysiological behavior, multipolar morphology, and double labeling with calbindin, which is a chemical code for AH-type neurons in the guinea pig ENS. Bradykinin evoked depolarizing responses in the AH-type neurons and the depolarization was associated with increased neuronal input resistance and reduction in amplitude of the characteristic long-lasting hyperpolarizing after-potentials in these neurons. The mean reversal potential for the depolarizing responses to BK in AH-type neurons was -92 mV and was characteristic of the depolarizing responses reported for the action of other excitatory mediators (e.g., histamine, substance P, and serotonin) on AH-type neurons (Katayama and North, 1978; Wood and Mayer, 1979; Nemeth et al., 1984). The reversal potential of -92 mV in AH-type neurons was close to the estimated K⁺ equilibrium potential and suggestive of decreased K⁺ conductance as the ionic mechanism underlying the depolarizing responses. Suppression of the hyperpolarizing after-potenials in AH-type neurons by BK was also characteristic for the action reported for other excitatory mediators, including substance P, histamine, and serotonin (Katayama and North, 1978; Wood and Mayer, 1979; Nemeth et al., 1984).

Bradykinin seems not to be an excitatory neurotransmitter in the submucosal plexus because there is no evidence that BK is stored and released by the ganglion cells. On the other hand, BK-containing neurons were reported to be present in the central nervous system (Correa et al., 1979). Our evidence overall suggests that BK behaves solely as a paracrine mediator in the ENS with excitatory actions on both S- and AH-type neurons in the submucosal plexus. The S-type neurons are primarily secretomotor neurons that receive excitatory synaptic input from their AH-type neighbors (Cooke, 2000). Enhanced excitability in both populations of submucosal neurons by BK is a mechanistic explanation for reports of stimulated mucosal secretion during exposure to BK (Kachur et al., 1987; Gaginella and Kachur, 1989). The pathophysiological significance of the neuronal actions of BK can therefore be related to the secretory diarrhea associated with intestinal inflammatory states in which the BK levels increase and the BK B₂ receptor is overexpressed (Stadnicki et al., 1998).

	Footnotes

 
This study was supported by National Institutes of Health Grant RO1 DK 57075. Prof. A. Müller-Esterl supplied AS276 as a generous gift.

DOI: 10.1124/jpet.103.059188.

ABBREVIATIONS: ENS, enteric nervous system; BK, bradykinin; RT-PCR, reverse transcription-polymerase chain reaction; PBS, phosphate-buffered saline; ChAT, choline acetyltransferase; NPY, neuropeptide Y; SP, substance P; VIP, vasoactive intestinal peptide; I-V, current-voltage; HOE-140, icatabant acetate; HOE-741, fluorescently labeled HOE-140; WIN64338 (S)-4[2-bis(cyclohexylamino)methyleneamino]-3-(2-napthalenyl)-1-oxopropylamino]benzyl tributyl phosphonium chloride hydrochloride.

Address correspondence to: Dr. Jackie D. Wood, Department of Physiology and Cell Biology, College of Medicine and Public Health, The Ohio State University, 304 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210-1218. E-mail: wood.13{at}osu.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Bachvarov DR, Hess JF, Menke JG, Larrivee JF, and Marceau F (1996) Structure and genomic organization of the human B1 receptor gene for kinins (BDKRB1). Genomics 33: 374-381.[CrossRef][Medline]

Bhoola KD, Figueroa CD, and Worthy K (1992) Bioregulation of kinins: kallikreins, kininogens and kininases. Pharmacol Rev 44: 1-80.[Medline]

Borkowski JA, Ransom RW, Seabrook GR, Trumbauer M, Chen H, Hill RG, Strader CD, and Hess JF (1995) Targeted disruption of a B₂ bradykinin receptor gene in mice eliminates bradykinin action in smooth muscle and neurons. J Biol Chem 270: 13706-13710.[Abstract/Free Full Text]

Bornstein JC and Furness JB (1988) Correlated electrophysiological and histochemical studies of submucous neurons and their contribution to understanding enteric neural circuits. J Auton Nerv Syst 25: 1-13.[CrossRef][Medline]

Bornstein JC, Furness JB, and Kunze WAA (1994) Electrophysiological characterization of myenteric neurons - how do classification schemes relate. J Auton Nerv Syst 48: 1-15.[CrossRef][Medline]

Brookes SJH (2001a) Classes of enteric nerve cells in the guinea-pig small intestine. Anat Rec 262: 58-70.[CrossRef][Medline]

Brookes SJH (2001b) Retrograde tracing of enteric neuronal pathways. Neurogastroenterol Motil 13: 1-18.[CrossRef][Medline]

Brown NJ and Roberts LJ (2001) Histamine, bradykinin and their antagonists, in The Pharmacological Basis of Therapeutics (Hardman JG, Limbird LE, and Gilman AG eds) pp 645-667, McGraw-Hill Companies, New York.

Cooke HJ (1994) Neuroimmune signaling in regulation of intestinal ion transport. Am J Physiol 266: G167-G178.

Cooke HJ (2000) Neurotransmitters in neuronal reflexes regulating intestinal secretion. Ann NY Acad Sci 915: 77-80.[Abstract/Free Full Text]

Correa FMA, Innis B, Uhl GR, and Snyder SH (1979) BK-like immunoreactive neuronal systems localized histochemically in rat brain. Proc Natl Acad Sci USA 76: 1489-1493.[Abstract/Free Full Text]

Dekkers JA, Akkermans LM, and Kroese AB (1997) Effects of the inflammatory mediator prostaglandin E2 on myenteric neurons in guinea pig ileum. Am J Physiol 272: G1451-G1456.

Dray A and Perkins M (1993) Bradykinin and inflammatory pain. Trends Neurosci 16: 99-104.[CrossRef][Medline]

Farmer SG, Powell SJ, Wilkins DE, and Graham A (1998) Cloning, sequencing and functional expression of a guinea pig lung bradykinin B₂ receptor. Eur J Pharmacol 346: 291-298.[CrossRef][Medline]

Frieling T, Cooke HJ, and Wood JD (1991) Electrophysiological properties of neurons in submucous ganglia of the guinea-pig distal colon. Am J Physiol 260: G835-G841.

Frieling T, Rupprecht C, Kroese ABA, and Schemann M (1994) Effects of the inflammatory mediator prostaglandin D2 on submucosal neurons and secretion in guinea-pig colon. Am J Physiol 29: G132-G139.

Frieling T, Wood JD, and Cooke HJ (1992) Submucosal reflexes: distension-evoked ion secretion in the guinea pig distal colon. Am J Physiol 263: G91-G96.

Gaginella TS and Kachur JF (1989) Kinins as mediators of intestinal secretion. Am J Physiol 256: G1-G15.

Gao C, Liu S, Hu HZ, Gao N, Kim GY, Xia Y, and Wood JD (2002) Serine proteases excite myenteric neurons through protease-activated receptors in guinea pig small intestine. Gastroenterology 123: 1554-1564.[CrossRef][Medline]

Gershon MD (1998) The Second Brain. Harper-Collins Publishers, New York.

Hess JF, Borkowski JA, Young GS, Strader CD, and Ransom RW (1992) Cloning and pharmacological characterization of a human bradykinin (BK-2) receptor. Biochem Biophys Res Commun 184: 260-268.[CrossRef][Medline]

Hu HZ, Gao N, Lin Z, Gao C, Liu S, Ren J, Xia Y, and Wood JD (2002) Chemical coding and electrophysiology of enteric neurons expressing neurofilament 145 in guinea-pig gastrointestinal tract. J Comp Neurol 442: 189-203.[CrossRef][Medline]

Hu HZ, Liu S, Gao N, Xia Y, Mostafa R, Ren J, Zafirov DH, and Wood JD (2003) Actions of bradykinin on electrical and synaptic behavior of neurones in the myenteric plexus of guinea-pig small intestine. Br J Pharmacol 138: 1221-1232.[CrossRef][Medline]

Kachur JF, Allbee W, Danho W, and Gaginella TS (1987) Bradykinin receptors: functional similarities in guinea pig gut muscle and mucosa. Regul Pept 17: 63-69.[CrossRef][Medline]

Katayama Y and North RA (1978) Does substance P mediate slow synaptic excitation within the myenteric plexus? Nature (Lond) 274: 387-388.[CrossRef][Medline]

Kunze WAA and Furness JB (1999) The enteric nervous system and regulation of intestinal motility. Annu Rev Physiol 61: 117-142.[CrossRef][Medline]

Lin Z, Na G, Hu HZ, Liu S, Gao C, Kim G, Ren J, Xia Y, Peck OC, and Wood JD (2002) Immunoreactivity of Hu proteins facilitates identification of myenteric neurons in guinea-pig small intestine. Neurogastroenterol Motil 14: 197-204.[CrossRef][Medline]

Liu S, Hu HZ, Gao C, Gao N, Wang G, Wang X, Gao X, Xia Y, and Wood JD (2003b) Actions of cysteinyl leukotrienes in the enteric nervous system of guinea-pig stomach and small intestine. Eur J Pharmacol 459: 27-39.[CrossRef][Medline]

Liu S, Hu HZ, Gao N, Gao C, Wang G, Wang X, Peck OC, Kim G, Gao X, Xia Y, and Wood JD (2003a) Neuroimmune interactions in guinea pig stomach and small intestine. Am J Physiol 284: G154-G164.

Ma JX, Wang DZ, Chao L, and Chao J (1994) Cloning, sequence analysis and expression of the gene encoding the mouse bradykinin B₂ receptor. Gene 149: 283-288.[CrossRef][Medline]

McEachern AE, Shelton ER, Bhakta S, Obernolte R, Bach, Zuppan P, Fijisaka J, Aldrich RW, and Jarjagin K (1991) Expression cloning of a rat B₂ bradykinin receptor. Proc Natl Acad Sci USA 88: 7724-7728.[Abstract/Free Full Text]

Nemeth PR, Ort CA, and Wood JD (1984) Intracellular study of effects of histamine on electrical behaviour of myenteric neurones in guinea-pig small intestine. J Physiol (Lond) 355: 411-425.[Abstract/Free Full Text]

North RA (1973) The calcium-dependent slow after-hyperpolarization in myenteric plexus neurones with tetrodotoxin-resistant action potentials. Br J Pharmacol 49: 709-711.[Medline]

Pruneau D, Luccarini JM, Defrene E, Paquet JL, and Belichard P (1995) Pharmacological evidence for a single bradykinin B₂ receptor in the guinea-pig. Br J Pharmacol 116: 2106-2112.[Medline]

Reyes-Cruz G, Vázquez-Prado J, Müller-Esterl W, and Vaca L (2000) Regulation of the human bradykinin B₂ receptor expressed in sf21 insect cells: a possible role for tyrosine kinases. J Cell Biochem 76: 658-673.[CrossRef][Medline]

Seabrook GR, Bowery BJ, Heavens R, Brown N, Ford H, Sirinathsinghi DJ, Borkowski JA, Hess JF, Strader CD, and Hill RG (1997) Expression of B₁ and B₂ bradykinin receptor mRNA and their functional roles in sympathetic ganglia and sensory dorsal root ganglia neurones from wild-type and B2 receptor knockout mice. Neuropharmacology 36: 1009-1017.[CrossRef][Medline]

Shen KZ and Surprenant A (1993) Common ionic mechanisms of excitation by substance P and other transmitters in guinea-pig submucosal neurones. J Physiol (Lond) 462: 483-501.[Abstract/Free Full Text]

Smith TK, Bornstein JC, and Furness JB (1990) Distension-evoked ascending and descending reflexes in the circular muscle of guinea-pig ileum: an intracellular study. J Auton Nerv Syst 29: 203-217.[CrossRef][Medline]

Stadnicki A, Sartor RB, Janardham R, Stadnicka I, Adam AA, Blais C Jr, and Colman RW (1998) Kallikrein-kininogen system activation and bradykinin (B2) receptors in indomethacin induced enterocolitis in genetically susceptible Lewis rats. Gut 43: 365-374.[Abstract/Free Full Text]

Wood JD (1994a) Physiology of the enteric nervous system, in Physiology of the Gastrointestinal Track, 3rd ed. (Johnson LR, Alpers DA, Christensen J, Jacobson ED, and Walsh JA eds) pp 423-482, Raven Press, New York.

Wood JD (1994b) Application of classification schemes to the enteric nervous system. J Auton Nerv Syst 48: 17-29.[CrossRef][Medline]

Wood JD (2002) Enteric neuro-immuno physiology, in Innervation of the Gastrointestinal Tract (Brookes SJH, Costa M, and Burnstock G eds) pp 363-392, Taylor & Francis Ltd., London.

Wood JD, Alpers DH, and Andrews PL (1999) Fundamentals of neurogastroenterology. Gut 45 (Suppl 2): 6-16.[Free Full Text]

Wood JD and Mayer CJ (1979) Serotonergic activation of tonic-type enteric neurons in guinea pig small bowel. J Neurophysiol 42: 582-593.[Abstract/Free Full Text]

Xia Y, Hu H-Z, Liu S, Ren J, Zafirov DH, and Wood JD (1999) IL-1B and IL-6 excite neurons and suppress nicotinic and noradrenergic neurotransmission in guinea pig enteric nervous system. J Clin Investig 103: 1309-1316.[Medline]

Zeitlin IJ and Smith AN (1973) Mobilization of tissue kallikrein in inflammatory disease of the colon. Gut 14: 133-138.[Abstract/Free Full Text]

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