Proc. Nati. Acad. Sci. USAVol. 91, pp. 4412-4416, May 1994Neurobiology

Identification of a B2 bradykinin receptor expressed by PC12pheochromocytoma cells

(chromaffin cell/sensory neuron/peripheral nervous system/pain/iammaton)

JULIE NARDONE*, CHRISTOPHE GERALDt, LAMA RIMAWI, Lucy SONG, AND PATRICK G. HOGANtDepartment of Neurobiology, Harvard Medical School, Boston, MA 02115

Communicated by Edwin J. Furshpan, January 7, 1994

ABSTRACT We have used rat PC12 pheochromocytomacels, a clonal cell flue closely related to sympathetic neurons, toInvesdite reports that the bradykinin receptor expressed In theperipheral nervous system Is distinctfomthe well-characterizedB2 bradykinin receptor of smooth muscle. Although there havebeen reports that [Thl5',D-Phe7bradyklinn [where Thi is J-(2-thienyl)alaninel is a full agonist at some sites In the peripheralnervous system, we find that in PC12 cells [This",D-Phe7Jbradykinin behaves as a competitive antagonist of brady-kinin-stimulated phosphatidylinositol turnover. In particular,ufficint concentrations of [ThI5",D-Phe7jbradykinin com-

pletely block the increase in inositol bisphosphate and tisphos-phate in response to 100 nM bradykinin; [ThlS8,D-Phe7bradykinin alone, at up to 10 FM, does not appreciablyincrease inositol bisphosphate and trisphosphate. In contrast tothe absence of evidence for a distinctive neuronal receptor, wehave found convincing evidence that the bradykinin receptorpreviously identified In smooth muscle is present in PC12 cells.Using the polymerase chain reaction, we have isolated a full-length cDNA encoding a bradykinin receptor that is expressedin PC12 cells and verified that its nuleotide sequence is identicalexcept at a single position to that ofthe rat uterine B2 bradykininreceptor. When expressed in COS cells this uterine bradykininreceptor exhibits the same high affinity for [3Hjbradykinin (Kd4.4 uM), the same relative affnities for a series of kininantagonists, and the same efficient coupling to phophatidylino-sitol turnover (ECso 2.5 nM) as the receptor in PC12 cells. Weinterpret our data, and the ings of a number of pharmacological studies, as strengthe the view that the B2 receptorexpressed in PC12 cells and in certain cells of the peripheralnervous system is identical to the receptor in rat uterine smoothmuscle.

The inflammatory nonapeptide bradykinin has a variety ofdirect and indirect effects on cells of the peripheral nervoussystem. Bradykinin excites nociceptive sensory endings (1-5), producing a sensation of pain (6, 7). Chemical mediatorsreleased by bradykinin from sensory endings (8-10) and fromsympathetic nerve terminals (11) contribute to vasodilatationand plasma leakage at the site of inflammation. Further, aspart of its inflammatory effect, bradykinin triggers the pro-duction of prostaglandin E or other arachidonic acid metab-olites in some tissues, and these mediators secondarily causea sensitization of sensory endings to bradykinin and otherstimuli (12, 13).

Bradykinin acts directly on neurons or nerve terminals incertain cases. Isolated sensory neurons show the same in-tense firing in response to bradykinin as sensory nerve fibersin vivo (14) and show a corresponding depolarizing ioniccurrent (15-17), indicating that the neurons themselves ex-press the bradykinin receptor that mediates excitation. Com-

plementary evidence that sensory neurons express bradyki-nin receptors has been obtained by autoradiographic local-ization oflabeled bradykinin bound to histological sections ofdorsal root ganglia (18) and by measurement of [3H]brady-kinin binding to intact dorsal root ganglion cells (L.S. andP.G.H., unpublished work). Less direct evidence from stud-ies with explanted sympathetic ganglia and ganglion homoge-nates suggests that a principal inflammatory mediator ofhyperalgesia, prostaglandin E, is released by a direct actionof bradykinin on sympathetic neurons (19, 20), consistentwith the observation that bradykinin fails to elicit hyperal-gesia in the rat paw after sympathetic denervation (21).Preliminary evidence also has been obtained for 3H-bradykinin binding to intact sympathetic neurons (J.N. andP.G.H., unpublished work).Although a bradykinin receptor cDNA has been isolated

from rat uterine smooth muscle (22) and from human fibro-blasts (23), there is a continuing controversy as to whether adistinct neuronal form of the bradykinin receptor exists.Evidence from two pharmacological studies has been cited asdefining a distinctive neuronal bradykinin receptor, charac-terized by its full activation with the kinin analogue [Thi5'8,D-Phe7]bradykinin [where Thi is -Q(2-thienyl)alanine] (24, 25).However, these studies have been criticized on the groundsthat they did not demonstrate that the responses were receptormediated (26, 27) and that a partial agonist like [Thi5'8,D-Phe7]bradykinin may appear as a full agonist in some assayconditions (28). The question ofwhethera distinct neuronal B2receptor exists needs to be resolved, since such a receptorwould be an important pharmacological target.Here we show that there is no evidence for expression of

a distinct neuronal bradykinin receptor activated by[Thi5'8,D-Phe7]bradykinin in PC12 pheochromocytoma cells(29), a cell line derived from the peripheral nervous system.Rather these cells express the known smooth muscle form ofthe bradykinin receptor. A review of the available pharma-cological evidence also suggests that the bradykinin receptorinitially identified in smooth muscle may account for most oreven all the actions of bradykinin in the peripheral nervoussystem.

METHODSCell Culture. PC12 cells were maintained in L15CO2 me-

dium (30) containing 7% horse serum and 7% fetal bovineserum and supplemented with 16.7 mM glucose, penicillin(100 units/ml), streptomycin sulfate (100 pg/ml), fresh vita-min mix (30), and 2 mM glutamine. COS-1 cells (simian

Abbreviations: IP2, inositol bisphosphate; IP3, inositol trisphos-phate; Thi, f-(2-thienyl)alanine.*Present address: Department of Molecular Biology, MassachusettsGeneral Hospital, Boston, MA 02114.

tPresent address: Synaptic Pharmaceutical Corporation, Paramus,NJ 07652.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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virus-transformed monkey kidney cells) were maintained inL15CO2 medium containing 7.5% fetal bovine serum and thesame supplements. Cultures were grown at 37TC in a 5% CO2atmosphere.Polymerase Chain Reaction (PCR). PC12 cells were treated

with 7S nerve growth factor (1 pug/ml) for 24 hr, RNA wasisolated and selected on oligo(dT)-cellulose, and double-stranded cDNA was prepared from 1 pg of poly(A)+ RNAwith oligo(dT) as primer. Degenerate oligodeoxynucleotideprimers 5'-GCGCGAATTCCTGCAGATGTTIAAHATYA-CIACNCA-3' and 5'-GCAGCGAATTCAAYTGYTTRT-TICCIGCCCAST-3' (where I is deoxyinosine), correspond-ing to the amino and carboxyl termini of the rat uterine B2bradykinin receptor (22), with extensions that included re-striction enzyme recognition site and G-C clamp sequences,were used to prime amplification ofthe cDNA under standardPCR conditions (31). The products of this reaction wereseparated by agarose gel electrophoresis, and the 1l.2-kbfragment was recovered from the gel and again subjected toPCR with the same primers. The product of this secondreaction was digested with EcoRI and cloned into pGEM-7(Promega) for sequencing.

In further PCR experiments nondegenerate oligonucleo-tide primers based on the sequence of the rat uterine brady-kinin receptorcDNA were used to amplify PC12 cDNA madefrom poly(A)+ RNA, as just described, or to amplify single-stranded cDNA prepared from PC12 cell total RNA bypriming with an antisense oligonucleotide representing nu-cleotides 1018-999. The numbering throughout this papercorresponds to that of the rat uterine bradykinin receptor,GenBank accession no. M59967.cDNA Sequence Determination. The cDNA in pGEM-7 was

sequenced on both strands by the dideoxy chain-terminationmethod (32) using T7 and SP6 oligonucleotide primers andspecific sense and antisense oligonucleotide primers. PooledPCR product was prepared (33) and sequenced by the dide-oxy chain-termination method.

Expression in COS-1 Cells. The receptor cDNA was ex-cised from pGEM-7 by digestion with Xba I and HindIII andsubcloned into the expression vector pLGP3 (available fromthe authors). Since the cDNA sequence had shown that thesame receptor gene was expressed in PC12 cells as in ratuterine smooth muscle, three apparent Taq polymerase er-rors were corrected on the basis of the published sequence ofthe smooth muscle receptor, and a stop codon was addedimmediately after the codon for Gln366, by site-directedmutagenesis (34). After subcloning and mutagenesis thesequence of the entire cDNA insert was verified. The resultsdescribed in the text are for expression of this form of thereceptor. Later sequence analysis, described in Results,showed that one nucleotide in fact differs between the PC12bradykinin receptor cDNA and the rat uterine bradykininreceptor cDNA, resulting in a codon for alanine rather thanglycine at position 76 in the PC12 bradykinin receptor. Apreliminary comparison showed that receptors with eitherAla76 or Gly76 exhibited high-affinity binding of bradykinin,consistent with the presence of alanine at this position in thehuman fibroblast bradykinin receptor.The receptor cDNA was introduced into COS-1 cells by

electroporation (35). COS cells (2 x 106) were removed fromculture flasks by treatment with trypsin, suspended in asolution containing 20 pg of plasmid DNA, treated with a1600-V 0.5-msec pulse, diluted into medium, and returned tothe incubator.Ligand Binding Assays. Binding assays were performed on

replicate wells of COS cells (initial density, 1-25 x 104 cellsper well) in 24-well Linbro plates that had been precoatedwith poly(D-lysine). In preparation for the assay, the plateswere placed in an ice/water bath and preequilibrated with 2ml ofice-cold assay buffer (137.5 mM NaCl/5 mM KCI/2mM

MgCl2/1 mM CaCl2/10 mM glucose/10 mM Hepes, pH7.4/0.1% bovine serum albumin) containing protease inhib-itors (10 uM captopril, 5 mM bacitracin, 1 mM iodoaceticacid, and 1 mM 1,10-phenanthroline). The binding reactionwas initiated by replacing the buffer with 0.3 ml of the samebuffer containing [3H]bradykinin (DuPont/NEN; 102 Ci/mmol; 1 Ci = 37 GBq). After at least 90 min at 0C, the cellswere washed three times with 2 ml of buffer and weresolubilized in 1% sodium dodecyl sulfate. Bound radioactiv-ity was measured by liquid scintillation counting. All datapoints were determined in duplicate. Nonspecific bindingwas measured in the presence of20 ttM unlabeled bradykininand, in COS cell assays, never exceeded 1.3% oftotal bindingfor concentrations of [3H]bradykinin 20 nM or lower. Inexperiments where a competing ligand was present, thecompeting ligand was added at the same time as [3H]brady-kini.The same protocol was used to examine the affinities of

D-Phe7-substituted kinins for PC12 cells at 0°C. A modifiedbinding assay-30 sec of incubation at =22°C in assay buffercontaining 1 mM 1,10-phenanthroline and 100 ,uM SQ20,881(L.S. and P.G.H., unpublished work)-was used to demon-strate binding of [Thi5'8,D-Phe7]bradykinin to PC12 cellsunder the more physiological conditions used for phospha-tidylinositol turnover measurements.

Phosphatidylinositol Turnover. Immediately after transfec-tion, COS cells were diluted to 105 cells per ml in mediumwith [3H]inositol (20 ,uCi/ml) and plated into 24-well plates asfor the binding assays. Two days later the cells were preequil-ibrated at =22°C with assay buffer containing 1 mM 1,10-phenanthroline, exposed to the indicated concentration ofbradykinin in the same buffer for 20 sec, and extracted with10% (wt/vol) trichloroacetic acid. Inositol bisphosphate (1P2)and inositol trisphosphate (1P3) levels in the acid-solubleextract were measured according to Berridge et al. (36, 37).In the chromatography, two 4-ml fractions were collectedwith each buffer (A-E) directly into scintillation vials, and theradioactivity was measured by liquid scintillation counting.

Phosphatidylinositol turnover in PC12 cells was deter-mined under the conditions of the 30-sec binding assay at=22°C, except that SQ20,881 was omitted. Where indicated,the competing ligand [Thi5'8,D-Phe7]bradykinin was added atthe same time as bradykinin.

RESULTS AND DISCUSSIONWe have made a detailed examination of the bradykininreceptor in PC12 pheochromocytoma cells with a combina-tion ofpharmacological and molecular biological approaches.The use of a neuronal cell line rather than excised tissue orneuronal primary cell cultures avoided any ambiguities in theconclusions due to the presence of nonneuronal cells thatexpress bradykinin receptors.Pharmacology. In our first experiments we asked whether

PC12 cells expressed a neuronal form of the receptor thatcould be recognized by its full activation by [Thi5'8,D-Phe7]bradykinin. We examined phosphatidylinositol turn-over because this second-messenger system is reported to befully activated by 1 ,uM [Thi5'8,D-Phe7]bradykinin in NlE-115neuroblastoma cells (25). [Thi5'8,D-Phe7]Bradykinin did notstimulate phosphatidylinositol turnover in PC12 cells whentested at concentrations of 1 ,uM or 10 ,uM (Table 1). In thesame experiments there was a clear increase in IP3 and IP2 inresponse to bradykinin (Table 1).

[Thi5'8,D-Phe7]Bradykinin bound to receptor sites duringthese experiments, since it competed with [3H]bradykinin forbinding to PC12 cells under the conditions of the phosphati-dylinositol turnover assay (Fig. 1). The IC50 for [Thi5'8,D-Phe7]bradykinin estimated from two experiments was 680 nM,and the analogue was capable of reducing bradykinin binding

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Table 1. Lack of effect of [Thi5'8,D-Phe7]bradykinin on levels ofIP2 and IP3 in PC12 cells

Fold increase

Treatment IP2 IP3Group 1

1 AM [Thi5'8,D-Phe7]Bradykinin 1.03 ± 0.09 1.01 ± 0.081 AM Bradykinin 3.64 ± 0.58 3.68 ± 1.19

Group 210 PM [Thi58,D-Phe7]Bradykinin 1.12 ± 0.34 0.94 ± 0.20100 nM Bradykinin 2.67 2.78

In each experimental group (1 ,uM or 10 AuM [Thi5'8,D-Phe7lbradykinin), identical samples of the labeled cells were testedwith bradykinin at the concentration indicated. Values are means +SEM for six separate determinations, except that values for 100 nMbradykinin are the means of duplicate determinations.

to close to background levels, indicating full occupancy ofbradykinin binding sites at 10 IM [Thi5'8,D-Phe7]bradykinin.Further evidence that the ligand was occupying the functionalbradykinin receptor sites without causing an increase in inosi-tol phosphate levels was obtained by examining the effect ofincreasing concentrations of [Thi5'8,D-Phe7]bradykinin on theturnover of phosphatidylinositol in response to 100 nM brady-kinin (Fig. 2). [Thi5'8,D-Phe7]Bradykinin at 1 ,uM or higherconcentrations diminished the levels of IP2 and IP3 relative tothe levels with 100 nM bradykinin alone. [Thi5'8,D-Phe7]Bradykinin at 100 ,uM blocked the response to bradyki-nin almost completely.These data indicate that a neuronal bradykinin receptor of

the type reported in peripheral sympathetic nerve terminalsand in NlE-ilS cells is not an appreciable contributor to theresponse ofPC12 cells. The results are not in conflict with thefinding that [Thi5'8,D-Phe7]bradykinin has very weak partialagonist activity at B2 receptors in many tissues, since ourassays would not resolve a very small increase in IP2 and IP3.cDNA Isolation and Sequence. Next we asked whether the

bradykinin receptor identified in rat smooth muscle (22) wasexpressed in PC12 cells. We prepared cDNA from PC12 cellsand subjected it to PCR using primers spanning the codingsequence of the rat uterine B2 receptor. An =1.2-kb PCRproduct was cloned into pGEM-7, sequenced, and found toencode the B2 receptor previously isolated from rat uterus.Of three nucleotide substitutions in this cloned PCR productrelative to the reported B2 receptor sequence, two wereattributable to incorporation of mismatched bases by Taq

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FIG. 1. Competition by [Thi5'8,D-Phe7lbradykinin for bradykininbinding sites in PC12 cells under physiological conditions.[3H]Bradykinin concentration was 16.3 nM, and IC50 was 520 nM inthe experiment shown. Arrowheads indicate the means of duplicatedeterminations of total [3H]bradykinin binding (T) and nonspecificbinding (N).

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FIG. 2. Antagonism of bradykinin-stimulated phosphatidylinosi-tol turnover by [Thi58,D-Phe7]bradykinin. Data represent bradyki-nin-stimulated increases in IP2 (a) and IP3 (b) in the presence ofvarious concentrations of [Thi5'8,D-Phe7]bradykinin, expressed as a

percentage of the increase caused by bradykinin alone. Bradykininconcentration was 100 nM in all experiments. The IC5o for IP2 was4.7 ,uM; and for IP3, 10 AM. The shift in IC50 compared to thatdetermined in Fig. 1 is largely explained by the higher bradykininconcentration, but a nonlinear relation between receptor occupancyand second-messenger production may also have contributed.

polymerase, since the substitutions were not observed whenfurther PCR products were sequenced. A single G -- C

substitution at nucleotide 606 was reproduced in independentPCRs, indicating that residue 76 of the PC12 bradykininreceptor is alanine. This single difference may representeither an allelic variant of the rat B2 receptor or a mutationthat has occurred in the PC12 cell line, but in either case thesame receptor gene is expressed in PC12 cells as in uterinesmooth muscle.

In view of evidence that the human B2 receptor gene doesnot have an intron in the coding sequence (28), it wasessential to eliminate the possibility that the cDNA we hadobtained by PCR arose from traces of genomic DNA con-taminating the RNA preparation. Therefore we carried outPCR amplifications with primers representing bases 122-141in the 5' untranslated region and bases 976-995 in the codingsequence of the rat uterine bradykinin receptor. The ex-pected -900-bp fragment was reproducibly obtained fromPC12 cDNA, but no product was obtained from PC12 RNAthat had been subjected to a mock cDNA synthesis reactionwith reverse transcriptase omitted. We verified by DNAsequencing of the total PCR products that the amplifiedproduct in fact encoded the expected fragment of the B2bradykinin receptor.We conclude that an mRNA is expressed in PC12 cells that

codes for a bradykinin receptor identical to that in smoothmuscle.

Binding Characteristics of the Cloned Receptor. To deter-mine whether this cDNA encoded a protein whose charac-teristics were consonant with those of the receptor in PC12cells, we expressed the receptor in COS-1 cells and studiedits binding of bradykinin and bradykinin antagonists. ThecDNA was subcloned into the COS cell expression vectorpLGP3 and introduced into COS-1 cells by electroporation.After 2 days, binding of [3H]bradykinin was examined in anequilibrium binding assay (Fig. 3). Binding increased and

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began to plateau as the concentration of [3H]bradykinin wasincreased in the low nanomolar range. The data were con-sistent with a single class of binding sites with Kd 4.4 + 1.6nM (n = 16; range, 1.5-7.5 nM). At saturating concentrationsof bradykinin, 1.1 ± 0.53 x 106 sites (n = 16; range, 2.9 x105-2.3 x 106) were occupied per cell transformed withcDNA. Control COS cells that were not transformed with thecDNA, and cells transformed with certain mutated cDNAs(38), showed no appreciable binding of [3H]bradykinin.The binding site was further characterized by measuring its

affinities for a series of D-Phe7-substituted kinin antagonists(Fig. 4 a-c). All of the peptides examined-D-Arg-[Hyp3,D-Phe7]bradykinin (where Hyp is hydroxyproline), [Thi5'8,D-Phe7]bradykinin, and [D-Phe7]Bradykinin--competed with[3H]bradykinin for binding to the site expressed in COS cells.The Kd values for binding these ligands were 7.5 ± 1.8 nM (n= 6), 130 ± 59 nM (n = 3), and 230 ± 61 nM (n = 3),respectively, giving the same order of affinities as that deter-mined for the B2 receptor in PC12 cells (Fig. 4d). Pharmaco-logically the receptor was a B2 bradykinin receptor, since itwas able to bind bradykinin and the D-Phe7-substituted kininswith high affinity and exhibited a strong preference for brady-kinin over the classical B1 agonist des-Arg9-bradykinin (Fig.4e).These results extend work with the rat uterine bradykinin

receptor cDNA (22) by measuring directly the interaction ofthe receptor with a series of kinin ligands. Our conclusionthat the cDNA encodes a B2 bradykinin receptor is inagreement with pharmacological data for the rat uterine B2receptor expressed in Xenopus oocytes (22).

Coupling of the Cloned Receptor to PhosphatidylinositolTurnover. Because a prominent action of bradykinin in PC12cells is to increase levels of inositol phosphates, we tested forcoupling of the receptor expressed in COS cells to thissecond-messenger pathway. Cells were prelabeled with[3H]inositol and stimulated with bradykinin for 20 sec. Max-imal increases in the inositol phosphates, in response to 1 JIMbradykinin, were 2.6 ± 0.42-fold for IP3 and 1.8 + 0.66-foldfor IP2 (n = 4). In control COS cells there was no stimulationof inositol phosphate production by bradykinin. In twofurther experiments that examined stimulation by bradykininin more detail, bradykinin at nanomolar concentrationssharply increased IP2 and IP3. In preliminary quantitative

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FIG. 3. [3H]Bradykinin binding to COS cells expressing thecloned bradykinin receptor. (a) e, Total binding; o, nonspecificbinding. In the same range of [3H]bradykinin concentrations, specificbinding to control COS cells not transformed with the receptorcDNA was <100 cpm. (b) Scatchard analysis of specific binding dataindicates a Kd of 3.9 nM in this experiment.

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FIG. 4. Competition of selected kinins with [3H]bradykinin in anequilibrium binding assay. (a-c) Competition of D-Phe7-substitutedkinins for binding to COS cells expressing the cloned receptor. *,D-Arg-[Hyp3,D-Phe7]bradykinin; o, [Thi5'8,D-Phe7]bradykinin; *,

[D-Phe7]bradykinin. (d) Competition of D-Phe7-substituted kinins forbinding to PC12 cells. *, D-Arg-[Hyp3,D-Phe7]bradykinin; o,[Thi5'8,D-Phe7]bradykinin; *, [D-Phe7lbradykinin. The respective Kivalues calculated from two such experiments with PC12 cells were7.0, 180, and 180 nM. (e) Competition ofunlabeled bradykinin (o) anddes-Arg9-bradykinin (e) for binding to COS cells expressing thecloned receptor. Arrowheads at the left in each panel representbinding of [3H]bradykinin in the absence of competing ligand.Concentrations of [3H]bradykinin were 2.8 nM (a), 2.0 nM (b), 2.1nM (c), 2.5 nM (d), 2.0 nM (e).

experiments, the EC50 for increase in IP3 was 2.5 nM brady-kinin (n = 2; range, 1.5-3.5 nM). This is closely comparableto the EC50 of 15 nM observed in PC12 cells (L.S., L.R., andP.G.H., unpublished work), with the slightly greater sensi-tivity of COS cells to low concentrations of bradykininprobably due to the large number of receptors expressed percell.

Coupling of the receptor expressed in Xenopus oocytes tophosphatidylinositol turnover was inferred from the ability ofbradykinin to stimulate the electrophysiological responsecharacteristic of phosphatidylinositol turnover (22).B2 Receptor Type in Peripheral Neurons. The cDNA we

have isolated encodes a receptor that accounts for theprominent properties of the PC12 bradykinin receptor. Thereceptor binds the agonist [3H]bradykinin with high affinity(Kd 4.4 nM). D-Phe7-substituted kinin antagonists competewith [3H]bradykinin for binding to this site, but the specificB1 agonist des-Arg9-bradykinin does not compete effec-tively. Low nanomolar concentrations of bradykinin stimu-late phosphatidylinositol turnover through the receptor andpresumably also increase cytoplasmic Ca2+. While we cannotabsolutely rule out the presence ofother B2 receptors in PC12cells, the simplest interpretation of our results is that thissingle receptor accounts for all the effects of bradykinin onPC12 cells.A review of pharmacological studies at sensory nerve

terminals and at other sites in the peripheral nervous system

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indicates that, as in PC12 cells, [Thi5'8,D-Phe7]bradykinin isa blocker of bradykinin action in most of these preparations.[Thi5'8,D-Phe7]Bradykinin does not excite polymodal noci-ceptor sensory endings of dog spermatic nerve and inhibitsthe excitatory action of bradykinin on these sensory endings(39). In this instance the peptide is acting selectively as anantagonist ofbradykinin, since the responses to noxious heatand to stimulation with increased K+ are unaffected (39).Similarly, Griesbacher and Lembeck (40) state that thisanalogue inhibits excitation of nociceptors in rabbit auricularnerve in response to bradykinin. Sengupta et al. (41) havefound a complex pharmacology for the excitation of afferentfibers of opossum vagus and splanchnic nerves, which theyinterpret as reflecting an indirect action of bradykinin and[Thi5'8,D-Phe7]bradykinin on mechanoreceptors, due to theircontractile effect on esophageal smooth muscle; a directexcitation of nociceptive sensory endings by bradykinin; anda purely antagonistic action of [Thi5'8,D-Phe7]bradykinin atthe B2 receptor ofthe nociceptive sensory endings. [Thi5'8,D-Phe7]Bradykinin is also a blocker of bradykinin action onbovine adrenal chromaffin cells (42, 43). The only docu-mented case in which [Thi5'8,D-Phe7]bradykinin mimics theaction of bradykinin in the peripheral nervous system is thepresynaptic action on sympathetic nerve endings in the vasdeferens (24). For reasons discussed in the Introduction, it isnot possible to decide whether this result indicates thepresence of a distinct type of B2 receptor in sympatheticneurons.The possibility that the B2 receptor originally identified in

smooth muscle is expressed in the peripheral nervous systemmust be considered seriously. B2 receptors in the peripheralnervous system-at least in sensory neurons and adrenalchromaffin cells-respond to [hi58,D-Phe7]bradykinin in thesame way as the smooth muscle bradykinin receptor. B2receptors in PC12 cells, a cell line derived from the peripheralnervous system, respond similarly. The current work pro-vides evidence that PC12 cells express the known B2 brady-kinin receptor and that the presence of this receptor canexplain the prominent actions ofbradykinin on these cells. Toclarify further the molecular basis for the action ofbradykininin the peripheral nervous system, it will be necessary eitherto identify this receptor in rigorously purified sympatheticand sensory neurons and demonstrate that it is coupled totheir physiological responses or to identify another bradyki-nin receptor that is present in these neurons and accounts forthe responses.

This work was supported by grants from the National Institutes ofHealth (NS25078) and from the National Institute on Drug Abuse(DA05088 and DA04582). J.N. was supported by the Quan Fellow-ship at Harvard Medical School.

1. Beck, P. W. & Handwerker, H. 0. (1974) Pfldgers Arch. 347,209-222.

2. Kumazawa, T. & Mizumura, K. (1977) Brain Res. 136, 553-558.

3. Mense, S. (1977) J. Physiol. (London) 267, 75-88.4. Kaufman, M. P., Iwamoto, G. A., Longhurst, J. C. & Mitch-

ell, J. H. (1982) Circ. Res. 50, 133-139.5. Szolcs.nyi, J. (1987) J. Physiol. (London) 38, 9-23.6. Armstrong, D., Dry, R. M. L., Keele, C. A. & Markham,

J. W. (1953) J. Physiol. (London) 120, 326-351.7. Elliott, D. F., Horton, E. W. & Lewis, G. P. (1960) J. Physiol.

(London) 153, 473-480.8. Arvier, P. T., Chahl, L. A. & Ladd, R. J. (1977) Br. J. Phar-

macol. 59, 61-68.9. Butler, J. M. & Hammond, B. R. (1980) Br. J. Pharmacol. 69,

495-502.

10. Lundberg, J. M. & Saria, A. (1983) Nature (London) 302,251-253.

11. Coderre, T. J., Basbaum, A. I. & Levine, J. D. (1989) J.Neurophysiol. 62, 48-58.

12. Ferreira, S. H., Moncada, S. & Vane, J. R. (1973) Br. J.Pharmacol. 49, 86-97.

13. Levine, J. D., Lau, W., Kwiat, G. & Goetzl, E. J. (1984)Science 225, 743-745.

14. Baccaglini, P. I. & Hogan, P. G. (1983) Proc. Nati. Acad. Sci.USA 80, 594-598.

15. Burgess, G. M., Mullaney, I., McNeill, M., Dunn, P. M. &Rang, H. P. (1989) J. Neurosci. 9, 3314-3325.

16. McGehee,T). S. & Oxford, G. S. (1991) Mol. Cell. Neurosci. 2,21-30.

17. McGehee, D. S., Goy, M. F. & Oxford, G. S. (1992) Neuron 9,315-324.

18. Steranka, L. R., Manning, D. C., DeHaas, C. J., Ferkany,J. W., Borosky, S. A., Connor, J. R., Vavrek, R. J., Stewart,J. H. & Snyder, S. H. (1988) Proc. NatI. Acad. Sci. USA 85,3245-3249.

19. Gonzales, R., Goldyne, M. E., Taiwo, Y. 0. & Levine, J. D.(1989) J. Neurochem. 53, 1595-1598.

20. Suidan, H. S., Murrell, R. D. & Tolkovsky, A. M. (1991) Cell.Regul. 2, 13-25.

21. Levine, J. D.,Taiwo, Y. O., Collins, S. D.& Tam, J. K. (1986)Nature (London) 323, 158-160.

22. McEachern, A. E., Shelton, E. R., Bhakta, S., Obernolte, R.,Bach, C., Zuppan, P., Fujisaki, J., Aldrich, R. W. & Jarnagin,K. (1991) Proc. Natl. Acad. Sci. USA 88, 7724-7728.

23. Hess, J. F., Borkowski, J. A., Young, G. S., Strader, C. D. &Ransom, R. W. (1992) Biochem. Biophys. Res. Commun. 184,260-268.

24. Llona, I., Vavrek, R., Stewart, J. & Huidobro-Toro, J. P.(1987) J. Pharmacol. Exp. Ther. 241, 608-614.

25. Braas, K. M., Manning, D. C., Perry, D. C. & Snyder, S. H.(1988) Br. J. Pharmacol. 94, 3-5.

26. Bathon, J. M. & Proud, D. (1991) Annu. Rev. Pharmacol.Toxicol. 31, 129-162.

27. Farmer, S. G. & Burch, R. M. (1992) Annu. Rev. Pharmacol.Toxicol. 32, 511-536.

28. Eggerickx, D., Raspe, E., Bertrand, D., Vassart, G. & Par-mentier, M. (1992) Biochem. Biophys. Res. Comntun. 187,1306-1313.

29. Greene, L. A. & Tischler, A. (1976) Proc. Natd. Acad. Sci.USA 73, 2424-2428.

30. Mains, R. E. & Patterson, P. H. (1973) J. Cell Biol. 59, 329-345.

31. Innis, M. A. & Gelfand, D. H. (1990) in PCR Protocols, eds.Innis, M. A., Gelfand, D. H., Sninsky, J. J. & White, T. J.(Academic, San Diego), pp. 3-12.

32. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natd.Acad. Sci. USA 74, 5463-5467.

33. Yandell, D. W. & Dryja, T. P. (1989) Am. J. Hum. Genet. 45,547-555.

34. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987) MethodsEnzymol. 154, 367-382.

35. Potter, H., Weir, L. & Leder, P. (1984) Proc. Natd. Acad. Sci.USA 81, 7161-7165.

36. Berridge, M. J., Dawson, R. M. C., Downes, P., Heslop, J. P.& Irvine, R. F. (1983) Biochem. J. 212, 473-482.

37. Berridge, M. J. (1983) Biochem. J. 212, 849-858.38. Nardone, J. & Hogan, P. G. (1994) Proc. Natl. Acad. Sci. USA

91, 4417-4421.39. Mizumura, K., Minagawa, M., Tsujii, Y. & Kumazawa, T.

(1990) Pain 40, 221-227.40. Griesbacher, T. & Lembeck, F. (1987) Br. J. Pharmacol. 92,

333-340.41. Sengupta, J. N., Saha, J. K. & Goyal, R. K. (1992) J. Neuro-

physiol. 68, 1053-1067.42. Owen, P. J., Plevin, R. & Boarder, M. R. (1989)J. Pharmacol.

Exp. Ther. 248, 1231-1236.43. Bunn, S. J., Marley, P. D. & Livett, B. G. (1990) Neuropep-

tides 15, 187-194.

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