Proc. Nati. Acad. Sci. USA Vol. 91, pp. 4412-4416, May 1994 Neurobiology Identification of a B2 expressed by PC12 pheochromocytoma cells (chromaffin cell/sensory neuron/peripheral nervous system/pain/iammaton) JULIE NARDONE*, CHRISTOPHE GERALDt, LAMA RIMAWI, Lucy SONG, AND PATRICK G. HOGANt Department of Neurobiology, Harvard Medical School, Boston, MA 02115 Communicated by Edwin J. Furshpan, January 7, 1994

ABSTRACT We have used rat PC12 pheochromocytoma plementary evidence that sensory neurons express bradyki- cels, a clonal cell flue closely related to sympathetic neurons, to nin receptors has been obtained by autoradiographic local- Invesdite reports that the expressed In the ization oflabeled bradykinin bound to histological sections of peripheral nervous system Is distinctfomthewell-characterized dorsal root ganglia (18) and by measurement of [3H]brady- B2 bradykinin receptor of . Although there have kinin binding to intact dorsal root ganglion cells (L.S. and been reports that [Thl5',D-Phe7bradyklinn [where Thi is J-(2- P.G.H., unpublished work). Less direct evidence from stud- thienyl)alaninel is a full agonist at some sites In the peripheral ies with explanted sympathetic ganglia and ganglion homoge- nervous system, we find that in PC12 cells [This",D- nates suggests that a principal inflammatory mediator of Phe7Jbradykinin behaves as a competitive antagonist of brady- hyperalgesia, prostaglandin E, is released by a direct action kinin-stimulated phosphatidylinositol turnover. In particular, of bradykinin on sympathetic neurons (19, 20), consistent ufficint concentrations of [ThI5",D-Phe7jbradykinin com- with the observation that bradykinin fails to elicit hyperal- pletely block the increase in inositol bisphosphate and tisphos- gesia in the rat paw after sympathetic denervation (21). phate in response to 100 nM bradykinin; [ThlS8,D- Preliminary evidence also has been obtained for 3H- Phe7bradykinin alone, at up to 10 FM, does not appreciably bradykinin binding to intact sympathetic neurons (J.N. and increase inositol bisphosphate and trisphosphate. In contrast to P.G.H., unpublished work). the absence of evidence for a distinctive neuronal receptor, we Although a bradykinin receptor cDNA has been isolated have found convincing evidence that the bradykinin receptor from rat uterine smooth muscle (22) and from human fibro- previously identified In smooth muscle is present in PC12 cells. blasts (23), there is a continuing controversy as to whether a Using the polymerase chain reaction, we have isolated a full- distinct neuronal form of the bradykinin receptor exists. length cDNA encoding a bradykinin receptor that is expressed Evidence from two pharmacological studies has been cited as in PC12 cells and verified that its nuleotide sequence is identical defining a distinctive neuronal bradykinin receptor, charac- except at a single position to thatofthe rat uterine B2 bradykinin terized by its full activation with the kinin analogue [Thi5'8,D- receptor. When expressed in COS cells this uterine bradykinin Phe7]bradykinin [where Thi is -Q(2-thienyl)alanine] (24, 25). receptor exhibits the same high affinity for [3Hjbradykinin (Kd However, these studies have been criticized on the grounds 4.4 uM), the same relative affnities for a series of kinin that they did not demonstrate that the responses were receptor antagonists, and the same efficient coupling to phophatidylino- mediated (26, 27) and that a partial agonist like [Thi5'8,D- sitol turnover (ECso 2.5 nM) as the receptor in PC12 cells. We Phe7]bradykinin may appear as a full agonist in some assay interpret our data, and the ings of a number of pharmaco conditions (28). The questionofwhethera distinct neuronal B2 logical studies, as strengthe the view that the B2 receptor receptor exists needs to be resolved, since such a receptor expressed in PC12 cells and in certain cells of the peripheral would be an important pharmacological target. nervous system is identical to the receptor in rat uterine smooth Here we show that there is no evidence for expression of muscle. a distinct neuronal bradykinin receptor activated by [Thi5'8,D-Phe7]bradykinin in PC12 pheochromocytoma cells The inflammatory nonapeptide bradykinin has a variety of (29), a cell line derived from the peripheral nervous system. direct and indirect effects on cells of the peripheral nervous Rather these cells express the known smooth muscle form of system. Bradykinin excites nociceptive sensory endings (1- the bradykinin receptor. A review of the available pharma- 5), producing a sensation of pain (6, 7). Chemical mediators cological evidence also suggests that the bradykinin receptor released by bradykinin from sensory endings (8-10) and from initially identified in smooth muscle may account for most or sympathetic nerve terminals (11) contribute to vasodilatation even all the actions of bradykinin in the peripheral nervous and plasma leakage at the site of inflammation. Further, as system. part of its inflammatory effect, bradykinin triggers the pro- duction of prostaglandin E or other arachidonic acid metab- METHODS olites in some tissues, and these mediators secondarily cause a sensitization of sensory endings to bradykinin and other Cell Culture. PC12 cells were maintained in L15CO2 me- stimuli (12, 13). dium (30) containing 7% horse serum and 7% fetal bovine Bradykinin acts directly on neurons or nerve terminals in serum and supplemented with 16.7 mM glucose, penicillin certain cases. Isolated sensory neurons show the same in- (100 units/ml), streptomycin sulfate (100 pg/ml), fresh vita- tense firing in response to bradykinin as sensory nerve fibers min mix (30), and 2 mM glutamine. COS-1 cells (simian in vivo (14) and show a corresponding depolarizing ionic current (15-17), indicating that the neurons themselves ex- Abbreviations: IP2, inositol bisphosphate; IP3, inositol trisphos- press the bradykinin receptor that mediates excitation. Com- phate; Thi, f-(2-thienyl)alanine. *Present address: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114. The publication costs ofthis article were defrayed in part by page charge tPresent address: Synaptic Pharmaceutical Corporation, Paramus, payment. This article must therefore be hereby marked "advertisement" NJ 07652. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 4412 Downloaded by guest on September 27, 2021 Neurobiology: Nardone et al. Proc. Natl. Acad. Sci. USA 91 (1994) 4413 virus-transformed monkey kidney cells) were maintained in MgCl2/1 mM CaCl2/10 mM glucose/10 mM Hepes, pH L15CO2 medium containing 7.5% fetal bovine serum and the 7.4/0.1% bovine serum albumin) containing protease inhib- same supplements. Cultures were grown at 37TC in a 5% CO2 itors (10 uM captopril, 5 mM bacitracin, 1 mM iodoacetic atmosphere. acid, and 1 mM 1,10-phenanthroline). The binding reaction Polymerase Chain Reaction (PCR). PC12 cells were treated was initiated by replacing the buffer with 0.3 ml of the same with 7S nerve growth factor (1 pug/ml) for 24 hr, RNA was buffer containing [3H]bradykinin (DuPont/NEN; 102 Ci/ isolated and selected on oligo(dT)-cellulose, and double- mmol; 1 Ci = 37 GBq). After at least 90 min at 0C, the cells stranded cDNA was prepared from 1 pg of poly(A)+ RNA were washed three times with 2 ml of buffer and were with oligo(dT) as primer. Degenerate oligodeoxynucleotide solubilized in 1% sodium dodecyl sulfate. Bound radioactiv- primers 5'-GCGCGAATTCCTGCAGATGTTIAAHATYA- ity was measured by liquid scintillation counting. All data CIACNCA-3' and 5'-GCAGCGAATTCAAYTGYTTRT- points were determined in duplicate. Nonspecific binding TICCIGCCCAST-3' (where I is deoxyinosine), correspond- was measured in the presence of20 ttM unlabeled bradykinin ing to the amino and carboxyl termini of the rat uterine B2 and, in COS cell assays, never exceeded 1.3% oftotal binding bradykinin receptor (22), with extensions that included re- for concentrations of [3H]bradykinin 20 nM or lower. In striction enzyme recognition site and G-C clamp sequences, experiments where a competing ligand was present, the were used to prime amplification ofthe cDNA under standard competing ligand was added at the same time as [3H]brady- PCR conditions (31). The products of this reaction were kini. separated by agarose gel electrophoresis, and the 1l.2-kb The same protocol was used to examine the affinities of fragment was recovered from the gel and again subjected to D-Phe7-substituted kinins for PC12 cells at 0°C. A modified PCR with the same primers. The product of this second binding assay-30 sec of incubation at =22°C in assay buffer reaction was digested with EcoRI and cloned into pGEM-7 containing 1 mM 1,10-phenanthroline and 100 ,uM SQ20,881 (Promega) for sequencing. (L.S. and P.G.H., unpublished work)-was used to demon- In further PCR experiments nondegenerate oligonucleo- strate binding of [Thi5'8,D-Phe7]bradykinin to PC12 cells tide primers based on the sequence of the rat uterine brady- under the more physiological conditions used for phospha- kinin receptor cDNA were used to amplify PC12 cDNA made tidylinositol turnover measurements. from poly(A)+ RNA, as just described, or to amplify single- Phosphatidylinositol Turnover. Immediately after transfec- stranded cDNA prepared from PC12 cell total RNA by tion, COS cells were diluted to 105 cells per ml in medium priming with an antisense oligonucleotide representing nu- with [3H]inositol (20 ,uCi/ml) and plated into 24-well plates as cleotides 1018-999. The numbering throughout this paper for the binding assays. Two days later the cells were preequil- corresponds to that of the rat uterine bradykinin receptor, ibrated at =22°C with assay buffer containing 1 mM 1,10- GenBank accession no. M59967. phenanthroline, exposed to the indicated concentration of cDNA Sequence Determination. The cDNA in pGEM-7 was bradykinin in the same buffer for 20 sec, and extracted with sequenced on both strands by the dideoxy chain-termination 10% (wt/vol) trichloroacetic acid. Inositol bisphosphate (1P2) method (32) using T7 and SP6 oligonucleotide primers and and inositol trisphosphate (1P3) levels in the acid-soluble specific sense and antisense oligonucleotide primers. Pooled extract were measured according to Berridge et al. (36, 37). PCR product was prepared (33) and sequenced by the dide- In the chromatography, two 4-ml fractions were collected oxy chain-termination method. with each buffer (A-E) directly into scintillation vials, and the Expression in COS-1 Cells. The receptor cDNA was ex- radioactivity was measured by liquid scintillation counting. cised from pGEM-7 by digestion with Xba I and HindIII and Phosphatidylinositol turnover in PC12 cells was deter- subcloned into the expression vector pLGP3 (available from mined under the conditions of the 30-sec binding assay at the authors). Since the cDNA sequence had shown that the =22°C, except that SQ20,881 was omitted. Where indicated, same receptor was expressed in PC12 cells as in rat the competing ligand [Thi5'8,D-Phe7]bradykinin was added at uterine smooth muscle, three apparent Taq polymerase er- the same time as bradykinin. rors were corrected on the basis ofthe published sequence of the smooth muscle receptor, and a stop codon was added immediately after the codon for Gln366, by site-directed RESULTS AND DISCUSSION mutagenesis (34). After subcloning and mutagenesis the We have made a detailed examination of the bradykinin sequence of the entire cDNA insert was verified. The results receptor in PC12 pheochromocytoma cells with a combina- described in the text are for expression of this form of the tion ofpharmacological and molecular biological approaches. receptor. Later sequence analysis, described in Results, The use of a neuronal cell line rather than excised tissue or showed that one nucleotide in fact differs between the PC12 neuronal primary cell cultures avoided any ambiguities in the bradykinin receptor cDNA and the rat uterine bradykinin conclusions due to the presence of nonneuronal cells that receptor cDNA, resulting in a codon for alanine rather than express bradykinin receptors. glycine at position 76 in the PC12 bradykinin receptor. A Pharmacology. In our first experiments we asked whether preliminary comparison showed that receptors with either PC12 cells expressed a neuronal form of the receptor that Ala76 or Gly76 exhibited high-affinity binding of bradykinin, could be recognized by its full activation by [Thi5'8,D- consistent with the presence of alanine at this position in the Phe7]bradykinin. We examined phosphatidylinositol turn- human fibroblast bradykinin receptor. over because this second-messenger system is reported to be The receptor cDNA was introduced into COS-1 cells by fully activated by 1 ,uM [Thi5'8,D-Phe7]bradykinin in NlE-115 electroporation (35). COS cells (2 x 106) were removed from neuroblastoma cells (25). [Thi5'8,D-Phe7]Bradykinin did not culture flasks by treatment with trypsin, suspended in a stimulate phosphatidylinositol turnover in PC12 cells when solution containing 20 pg of plasmid DNA, treated with a tested at concentrations of 1 ,uM or 10 ,uM (Table 1). In the 1600-V 0.5-msec pulse, diluted into medium, and returned to same experiments there was a clear increase in IP3 and IP2 in the incubator. response to bradykinin (Table 1). Ligand Binding Assays. Binding assays were performed on [Thi5'8,D-Phe7]Bradykinin bound to receptor sites during replicate wells of COS cells (initial density, 1-25 x 104 cells these experiments, since it competed with [3H]bradykinin for per well) in 24-well Linbro plates that had been precoated binding to PC12 cells under the conditions of the phosphati- with poly(D-lysine). In preparation for the assay, the plates dylinositol turnover assay (Fig. 1). The IC50 for [Thi5'8,D- were placed in an ice/water bath and preequilibrated with 2 Phe7]bradykinin estimated from two experiments was 680 nM, ml ofice-cold assay buffer (137.5 mM NaCl/5 mM KCI/2 mM and the analogue was capable of reducing bradykinin binding Downloaded by guest on September 27, 2021 4414 Neurobiology: Nardone et al. Proc. Natl. Acad Sci. USA 91 (1994)

Table 1. Lack of effect of [Thi5'8,D-Phe7]bradykinin on levels of A IP2 and IP3 in PC12 cells E 10,0o a Fold increase E 8110 Treatment IP2 IP3 61,O A co CO Group 1 0) 41 1 AM [Thi5'8,D-Phe7]Bradykinin 1.03 ± 0.09 1.01 ± 0.08 C.) C 21 1 AM Bradykinin 3.64 ± 0.58 3.68 ± 1.19 cm Group 2 l- 10 PM [Thi58,D-Phe7]Bradykinin 1.12 ± 0.34 0.94 ± 0.20 10 103 105 100 nM Bradykinin 2.67 2.78 08 In each experimental group (1 ,uM or 10 AuM [Thi5'8,D- Phe7lbradykinin), identical samples of the labeled cells were tested with bradykinin at the concentration indicated. Values are means + E 100 A b SEM for six separate determinations, except that values for 100 nM 80 bradykinin are the means of duplicate determinations. E A~~~~ IOR 60 to close to background levels, indicating full occupancy of bradykinin binding sites at 10 IM [Thi5'8,D-Phe7]bradykinin. a) 40 Further evidence that the ligand was occupying the functional 20 \ A an increase in inosi- a- A receptor sites without causing . bradykinin * .J. tol phosphate levels was obtained by examining the effect of 0 10 103 105 increasing concentrations of [Thi5'8,D-Phe7]bradykinin on the [Thi58,D-Phe7]Bradykinin, nM turnover ofphosphatidylinositol in response to 100 nM brady- kinin (Fig. 2). [Thi5'8,D-Phe7]Bradykinin at 1 ,uM or higher FIG. 2. Antagonism of bradykinin-stimulated phosphatidylinosi- concentrations diminished the levels of IP2 and IP3 relative to tol turnover by [Thi58,D-Phe7]bradykinin. Data represent bradyki- nin-stimulated increases in IP2 (a) and IP3 (b) in the presence of the levels with 100 nM bradykinin alone. [Thi5'8,D- various concentrations of [Thi5'8,D-Phe7]bradykinin, expressed as a Phe7]Bradykinin at 100 ,uM blocked the response to bradyki- percentage of the increase caused by bradykinin alone. Bradykinin nin almost completely. concentration was 100 nM in all experiments. The IC5o for IP2 was These data indicate that a neuronal bradykinin receptor of 4.7 ,uM; and for IP3, 10 AM. The shift in IC50 compared to that the type reported in peripheral sympathetic nerve terminals determined in Fig. 1 is largely explained by the higher bradykinin and in NlE-ilS cells is not an appreciable contributor to the concentration, but a nonlinear relation between receptor occupancy response ofPC12 cells. The results are not in conflict with the and second-messenger production may also have contributed. finding that has very weak partial [Thi5'8,D-Phe7]bradykinin polymerase, since the substitutions were not observed when agonist activity at B2 receptors in many tissues, since our would not resolve a very small increase in and IP3. further PCR products were sequenced. A single G -- C assays IP2 substitution at nucleotide 606 was reproduced in independent cDNA Isolation and Sequence. Next we asked whether the PCRs, indicating that residue 76 of the PC12 bradykinin bradykinin receptor identified in rat smooth muscle (22) was We cDNA from PC12 cells receptor is alanine. This single difference may represent expressed in PC12 cells. prepared either an allelic variant of the rat B2 receptor or a mutation and subjected it to PCR using primers spanning the coding that has occurred in the PC12 cell line, but in either case the sequence of the rat uterine B2 receptor. An =1.2-kb PCR same receptor gene is expressed in PC12 cells as in uterine product was cloned into pGEM-7, sequenced, and found to smooth muscle. encode the B2 receptor previously isolated from rat uterus. In view of evidence that the human B2 receptor gene does Of three nucleotide substitutions in this cloned PCR product not have an intron in the coding sequence (28), it was relative to the reported B2 receptor sequence, two were essential to eliminate the possibility that the cDNA we had attributable to incorporation of mismatched bases by Taq obtained by PCR arose from traces of genomic DNA con- taminating the RNA preparation. Therefore we carried out A PCR amplifications with primers representing bases 122-141 co in the 5' untranslated region and bases 976-995 in the coding 2. 0 T ex- x sequence of the rat uterine bradykinin receptor. The E pected -900-bp fragment was reproducibly obtained from PC12 cDNA, but no product was obtained from PC12 RNA 1..5 - that had been subjected to a mock cDNA synthesis reaction C with reverse transcriptase omitted. We verified by DNA 0 Q .0 1 sequencing of the total PCR products that the amplified ._ product in fact encoded the expected fragment of the B2 bradykinin receptor. X 0..5 -NB- We conclude that an mRNA is expressed in PC12 cells that m codes for a bradykinin receptor identical to that in smooth I, muscle. Binding Characteristics of the Cloned Receptor. To deter- 1 100 1,000 mine whether this cDNA encoded a protein whose charac- nM [Thi5'8,D-Phe7]Bradykinin, teristics were consonant with those of the receptor in PC12 FIG. 1. Competition by [Thi5'8,D-Phe7lbradykinin for bradykinin cells, we expressed the receptor in COS-1 cells and studied binding sites in PC12 cells under physiological conditions. its binding of bradykinin and bradykinin antagonists. The [3H]Bradykinin concentration was 16.3 nM, and IC50 was 520 nM in cDNA was subcloned into the COS cell expression vector the experiment shown. Arrowheads indicate the means of duplicate pLGP3 and introduced into COS-1 cells by electroporation. determinations of total [3H]bradykinin binding (T) and nonspecific After 2 days, binding of [3H]bradykinin was examined in an binding (N). equilibrium binding assay (Fig. 3). Binding increased and Downloaded by guest on September 27, 2021 Neurobiology: Nardone et al. Proc. Natl. Acad. Sci. USA 91 (1994) 4415

began to plateau as the concentration of [3H]bradykinin was increased in the low nanomolar range. The data were con- sistent with a single class of binding sites with Kd 4.4 + 1.6 nM (n = 16; range, 1.5-7.5 nM). At saturating concentrations co of bradykinin, 1.1 ± 0.53 x 106 sites (n = 16; range, 2.9 x 0T- 105-2.3 x 106) were occupied per cell transformed with 4- To, 808 cDNA. Control COS cells that were not transformed with the x cDNA, and cells transformed with certain mutated cDNAs E C)0- 2 j (38), showed no appreciable binding of [3H]bradykinin. 4IT_0 as 0000 The binding site was further characterized by measuring its a to~~~ Cl affinities for series of D-Phe7-substituted kinin antagonists (Fig. 4 a-c). All of the examined-D-Arg-[Hyp3,D- Phe7]bradykinin (where Hyp is hydroxyproline), [Thi5'8,D- Phe7]bradykinin, and [D-Phe7]Bradykinin--competed with cs [3H]bradykinin for binding to the site expressed in COS cells. 3 - To- The Kd values for binding these ligands were 7.5 ± 1.8 nM (n In0~ = 6), 130 ± 59 nM (n = 3), and 230 ± 61 nM (n = 3), respectively, giving the same order of affinities as that deter- 1o mined for the B2 receptor in PC12 cells (Fig. 4d). Pharmaco- x logically the receptor was a B2 bradykinin receptor, since it E was able to bind bradykinin and the D-Phe7-substituted kinins C with high affinity and exhibited a strong preference for brady- 6 O t@e.. 1 kinin over the classical B1 agonist des-Arg9-bradykinin (Fig. 0 co 4e). 22 8 These results extend work with the rat uterine bradykinin receptor cDNA (22) by measuring directly the interaction of -10-9 -6-5 the receptor with a series of kinin ligands. Our conclusion that the cDNA encodes a B2 bradykinin receptor is in log[ligand] agreement with pharmacological data for the rat uterine B2 FIG. 4. Competition of selected kinins with [3H]bradykinin in an receptor expressed in Xenopus oocytes (22). equilibrium binding assay. (a-c) Competition of D-Phe7-substituted Coupling of the Cloned Receptor to Phosphatidylinositol kinins for binding to COS cells expressing the cloned receptor. *,

Turnover. Because a prominent action ofbradykinin in PC12 D-Arg-[Hyp3,D-Phe7]bradykinin; o, [Thi5'8,D-Phe7]bradykinin; *, cells is to increase levels ofinositol phosphates, we tested for [D-Phe7]bradykinin. (d) Competition of D-Phe7-substituted kinins for coupling of the receptor expressed in COS cells to this binding to PC12 cells. *, D-Arg-[Hyp3,D-Phe7]bradykinin; o, second-messenger pathway. Cells were prelabeled with [Thi5'8,D-Phe7]bradykinin; *, [D-Phe7lbradykinin. The respective Ki values calculated from two such experiments with PC12 cells were [3H]inositol and stimulated with bradykinin for 20 sec. Max- 7.0, 180, and 180 nM. (e) Competition ofunlabeled bradykinin (o) and imal increases in the inositol phosphates, in response to 1 JIM des-Arg9-bradykinin (e) for binding to COS cells expressing the bradykinin, were 2.6 ± 0.42-fold for IP3 and 1.8 + 0.66-fold cloned receptor. Arrowheads at the left in each panel represent for IP2 (n = 4). In control COS cells there was no stimulation binding of [3H]bradykinin in the absence of competing ligand. of inositol phosphate production by bradykinin. In two Concentrations of [3H]bradykinin were 2.8 nM (a), 2.0 nM (b), 2.1 further experiments that examined stimulation by bradykinin nM (c), 2.5 nM (d), 2.0 nM (e). in more detail, bradykinin at nanomolar concentrations sharply increased IP2 and IP3. In preliminary quantitative experiments, the EC50 for increase in IP3 was 2.5 nM brady- kinin (n = 2; range, 1.5-3.5 nM). This is closely comparable co) to the EC50 of 15 nM observed in PC12 cells (L.S., L.R., and 0 8- a6 - P.G.H., unpublished work), with the slightly greater sensi- x 6- I tivity of COS cells to low concentrations of bradykinin E I- 0-1 probably due to the large number of receptors expressed per 0 cell. C: 4- S P Coupling of the receptor expressed in Xenopus oocytes to 0 Uj -I turnover was inferred the m I ' phosphatidylinositol from ability of 5 10 15 20 2. 5 bradykinin to stimulate the electrophysiological response [3H]Bradykinin, nM characteristic of phosphatidylinositol turnover (22). B2 Receptor Type in Peripheral Neurons. The cDNA we 1 2.5- have isolated encodes a receptor that accounts for the U) ° 2.0- Snb prominent properties of the PC12 bradykinin receptor. The CD receptor binds the agonist [3H]bradykinin with high affinity x 1.5- (Kd 4.4 nM). D-Phe7-substituted kinin antagonists compete , c 1.0- with [3H]bradykinin for binding to this site, but the specific - m 0 E 0.5 B1 agonist des-Arg9-bradykinin does not compete effec- 0 nn, I tively. Low nanomolar concentrations of bradykinin stimu- 0 5 10 late phosphatidylinositol turnover through the receptor and Specific binding, cpm x 10-3 presumably also increase cytoplasmic Ca2+. While we cannot absolutely rule out the presence ofother B2 receptors in PC12 FIG. 3. to COS cells the [3H]Bradykinin binding expressing cells, the simplest interpretation of our results is that this cloned bradykinin receptor. (a) e, Total binding; o, nonspecific receptor accounts for all the effects of on binding. In the same range of [3H]bradykinin concentrations, specific single bradykinin binding to control COS cells not transformed with the receptor PC12 cells. cDNA was <100 cpm. (b) Scatchard analysis of specific binding data A review of pharmacological studies at sensory nerve indicates a Kd of 3.9 nM in this experiment. terminals and at other sites in the peripheral nervous system Downloaded by guest on September 27, 2021 4416 Neurobiology: Nardone et al. Proc. Natl. Acad. Sci. USA 91 (1994) indicates that, as in PC12 cells, [Thi5'8,D-Phe7]bradykinin is 10. Lundberg, J. M. & Saria, A. (1983) Nature (London) 302, a blocker ofbradykinin action in most of these preparations. 251-253. [Thi5'8,D-Phe7]Bradykinin does not excite polymodal noci- 11. Coderre, T. J., Basbaum, A. I. & Levine, J. D. (1989) J. ceptor sensory endings of dog spermatic nerve and inhibits Neurophysiol. 62, 48-58. the excitatory action of bradykinin on these sensory endings 12. Ferreira, S. H., Moncada, S. & Vane, J. R. (1973) Br. J. In this instance the is as an Pharmacol. 49, 86-97. (39). acting selectively 13. Levine, J. D., Lau, W., Kwiat, G. & Goetzl, E. J. (1984) antagonist ofbradykinin, since the responses to noxious heat Science 225, 743-745. and to stimulation with increased K+ are unaffected (39). 14. Baccaglini, P. I. & Hogan, P. G. (1983) Proc. Nati. Acad. Sci. Similarly, Griesbacher and Lembeck (40) state that this USA 80, 594-598. analogue inhibits excitation of nociceptors in rabbit auricular 15. Burgess, G. M., Mullaney, I., McNeill, M., Dunn, P. M. & nerve in response to bradykinin. Sengupta et al. (41) have Rang, H. P. (1989) J. Neurosci. 9, 3314-3325. found a complex pharmacology for the excitation of afferent 16. McGehee,T). S. & Oxford, G. S. (1991) Mol. Cell. Neurosci. 2, fibers of opossum vagus and splanchnic nerves, which they 21-30. interpret as reflecting an indirect action of bradykinin and 17. McGehee, D. S., Goy, M. F. & Oxford, G. S. (1992) Neuron 9, [Thi5'8,D-Phe7]bradykinin on mechanoreceptors, due to their 315-324. contractile effect on esophageal smooth muscle; a direct 18. Steranka, L. R., Manning, D. C., DeHaas, C. J., Ferkany, excitation of J. W., Borosky, S. A., Connor, J. R., Vavrek, R. J., Stewart, nociceptive sensory endings by bradykinin; and J. H. & Snyder, S. H. (1988) Proc. NatI. Acad. Sci. USA 85, a purely antagonistic action of [Thi5'8,D-Phe7]bradykinin at 3245-3249. the B2 receptor ofthe nociceptive sensory endings. [Thi5'8,D- 19. Gonzales, R., Goldyne, M. E., Taiwo, Y. 0. & Levine, J. D. Phe7]Bradykinin is also a blocker of bradykinin action on (1989) J. Neurochem. 53, 1595-1598. bovine adrenal chromaffin cells (42, 43). The only docu- 20. Suidan, H. S., Murrell, R. D. & Tolkovsky, A. M. (1991) Cell. mented case in which [Thi5'8,D-Phe7]bradykinin mimics the Regul. 2, 13-25. action of bradykinin in the peripheral nervous system is the 21. Levine, J. D.,Taiwo, Y. O., Collins, S. D. & Tam, J. K. (1986) presynaptic action on sympathetic nerve endings in the vas Nature (London) 323, 158-160. deferens (24). For reasons discussed in the Introduction, it is 22. McEachern, A. E., Shelton, E. R., Bhakta, S., Obernolte, R., not to decide whether this result Bach, C., Zuppan, P., Fujisaki, J., Aldrich, R. W. & Jarnagin, possible indicates the K. (1991) Proc. Natl. Acad. Sci. USA 88, 7724-7728. presence of a distinct type of B2 receptor in sympathetic 23. Hess, J. F., Borkowski, J. A., Young, G. S., Strader, C. D. & neurons. Ransom, R. W. (1992) Biochem. Biophys. Res. Commun. 184, The possibility that the B2 receptor originally identified in 260-268. smooth muscle is expressed in the peripheral nervous system 24. Llona, I., Vavrek, R., Stewart, J. & Huidobro-Toro, J. P. must be considered seriously. B2 receptors in the peripheral (1987) J. Pharmacol. Exp. Ther. 241, 608-614. nervous system-at least in sensory neurons and adrenal 25. Braas, K. M., Manning, D. C., Perry, D. C. & Snyder, S. H. chromaffin cells-respond to [hi58,D-Phe7]bradykinin in the (1988) Br. J. Pharmacol. 94, 3-5. same way as the smooth muscle bradykinin receptor. B2 26. Bathon, J. M. & Proud, D. (1991) Annu. Rev. Pharmacol. in PC12 a line from Toxicol. 31, 129-162. receptors cells, cell derived the peripheral 27. Farmer, S. G. & Burch, R. M. (1992) Annu. Rev. Pharmacol. nervous system, respond similarly. The current work pro- Toxicol. 32, 511-536. vides evidence that PC12 cells express the known B2 brady- 28. Eggerickx, D., Raspe, E., Bertrand, D., Vassart, G. & Par- kinin receptor and that the presence of this receptor can mentier, M. (1992) Biochem. Biophys. Res. Comntun. 187, explain the prominent actions ofbradykinin on these cells. To 1306-1313. clarify further the molecular basis for the action ofbradykinin 29. Greene, L. A. & Tischler, A. (1976) Proc. Natd. Acad. Sci. in the peripheral nervous system, it will be necessary either USA 73, 2424-2428. to identify this receptor in rigorously purified sympathetic 30. Mains, R. E. & Patterson, P. H. (1973) J. Cell Biol. 59, 329- and sensory neurons and demonstrate that it is coupled to 345. their or to 31. Innis, M. A. & Gelfand, D. H. (1990) in PCR Protocols, eds. physiological responses identify another bradyki- Innis, M. A., Gelfand, D. H., Sninsky, J. J. & White, T. J. nin receptor that is present in these neurons and accounts for (Academic, San Diego), pp. 3-12. the responses. 32. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. 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