SYNAPSE 15148-57 (1993)

Autoradiographic Localization of [ 125 I-TYR?]-Bradykinin Receptor Binding

Sites in the Guinea Pig Spinal Cord PAUL0 LOPES, SATYABRATA KAR, CHRISTINE TOUSIGNANT, DOMENICO REGOLI,

RGMI QUIRION, AND RGJEAN COUTURE Department of Physiology, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada H3C 357

(P.L., R.C.); Douglas Hospital Research Centre, Department of Psychiatry, Faculty of Medicine, McGill University, Montreal, Quebec, Canada H4H l R 3 (S.K., R.Q.); Department of Pharmacology, Faculty of

Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J l H 5N4 (C.T., D.R.)

KEY WORDS

ABSTRACT The present study aimed to localize and characterize [1251-Tyr81-BK binding sites in all major segments of the guinea pig spinal cord using in vitro quantita- tive receptor autoradiography. [ '251-Tyr8]-BK specific binding sites were localized pre- dominantly in superficial layers of the dorsal horn, with lamina I1 depicting the highest labelling. The density of specific binding in laminae I and I11 was moderate, whereas in other areas, i.e., laminae IV-X, lower amounts of labelling were noticed. The B, receptor antagonists D-Arg[Hyp3,Thi5,D-Tic7,0ic8]-BK (Hoe 1401, D-Arg[Hyp3,D-Phe7,Leu81-BK, Tyro,D-Arg[Hyp3,D-Phe7,Leu8]-BK, D-Arg[Tyr3,D-Phe7,Leu8]-BK, D-Arg[Hyp2,Thi5x8,D- Phe7]-BK, D-Arg[Hyp3,Leu8]-BK and D-Arg[Hyp3,Gly6,Leu81-BK as well as unlabelled [Tyr8]-BK inhibited [1251-Tyr8]-BK binding with respective K, values of 0.04, 12.4, 23.4, 34.5,43.5,33.5,23.0, and 0.6 nM while B, related molecules (Tyr0,des-Arglo-kallidin and [Leu8]-des-Argg-BK) did not significantly inhibit [1251-Tyr81-BK binding up to micromolar concentrations. These results indicate that the specific [1251-Tyr8]-BK binding sites present in the guinea pig spinal cord belong to the B, receptor subtype. The high density of B2 binding sites in the substantia gelatinosa provides an anatomical evidence in favour of a role for BK as a modulator of nociceptive information.

Autoradiography, Bradykinin receptors, Dorsal horn, Motoneuron

o 1993 Wiley-Liss, Inc.

INTRODUCTION Bradykinin (BK), a nonapeptide produced during tis-

sue damage, is involved in inflammatory reactions causing pain by activation of peripheral sensory nerve endings, vasodilation and increased vascular perme- ability (Regoli and Barabe, 1980; Marceau et al., 1983). Under normal conditions, the majority of the biological effects of BK are mediated by the B, kinin receptor (Regoli and Barabe, 1980; Marceau et al., 1983; Regoli, 1984; Bhoola et al., 1992; Bathon and Proud, 1991), while the B, kinin receptor is generally synthesized de novo following tissue injury and in response to chemical noxious stimuli (Regoli and Barabe, 1980; Marceau et al., 1983). Des-Argg-BK and des-ArglO-kallidin are se- lective agonists of B, receptors, whereas B, receptors essentially do not respond to des-Argg-BK analogues (Regoli and Barabe, 1980). [Leu8]-des-Argg-BK is con- sidered as the prototype B, receptor antagonist while Hoe 140 (D-Arg-[Hyp3,Thi5,D-Tic7,0ics]-BK) is the most potent, long-acting and selective B, receptor an- tagonist described so far in vitro and in vivo (Wirth et al., 1991; Hock et al., 1991; Lembeck et al., 1991; 0 1993 WILEY-LISS, INC.

Rhaleb et al., 1992). Recently, several studies based on the inability of B, receptor antagonists to block BK- induced effects, have challenged the existence of only two BK receptors (Burch and Kyle, 1992; Braas et al., 1988; Rifo et al., 1987; Saha et al., 1989; Fujiwara et al., 1989; Rhaleb et al., 1991). Based on the activity of a series of agonists and antagonists, subtypes of B, recep- tors (B2* and B2B) have been proposed to be present in four isolated peripheral smooth muscle preparations (Regoli et al., 1992a). Subtypes of B, receptor binding sites have also been reported in intestinal epithelia, fibroblasts, primary brain cultures and in neuroblas- toma cell lines (Manning et al., 1986; Braas et al., 1988; Seguin et al., 1992; Burch and Kyle, 1992).

There is evidence that endogenous kinins, a t the level of the spinal cord, may play a role in the control of nociceptive information and on blood pressure (Laneu- ville et al., 1989; Lopes and Couture, 1992). BK-like

Received January 25,1993; accepted in revised form April 14,1993 Address correspondence to Rejean Couture.

49 BRADYKlNIN RECEPTORS IN THE GUINEA PIG SPINAL CORD

immunoreactivity has also been shown to be present in the rat spinal cord (Perry and Snyder, 1984). Function- ally, an initial nociceptive response followed by an anal- gesia is elicited by intrathecally administered BK in the awake rat (Laneuville and Couture, 1987; Laneuville et al., 1989). The nociception may be related to a direct action of BK on central sensory nerve terminals, as the presence of B, receptor binding sites in the substantia gelatinosa of the guinea pig spinal cord was reported (Steranka et al., 1988). This nociception, as expressed by behavioral excitation, is also consistent with the slow excitation of thermonociceptive dorsal horn neu- rons observed following iontophoretic application of BK in the cat spinal cord (Henry, 1976). On the other hand, the antinociception, as shown by a period of sedation and inhibition of thermonociception, was ascribed to an action of BK on bulbospinal noradrenergic inhibitory fibers (Laneuville et al., 1989). In addition, intrathecal administration of BK in the conscious rat increased blood pressure through the activation of the sym- pathoadrenal system. Actions of BK on spinal sensory terminals or on bulbospinal noradrenergic fibers were excluded as mechanisms likely to explain the pressor response to BK (Lopes and Couture, 1992). Hence, the possibility must be considered that BK acts directly on preganglionic sympathetic fibers in the intermediolat- era1 cell column of the spinal cord. However, the pres- ence of BK receptors in this area of the spinal cord as yet to be reported.

The present study was therefore undertaken to as- sess the detailed anatomical laminar distribution of BK receptor binding sites, in major segments of the guinea pig spinal cord using in vitro quantitative receptor au- toradiography. This study has been conducted in guinea pigs to permit comparison with earlier autorad- iographic (Steranka et al., 1988) and binding (Fujiwara et al., 1989; Sharif and Whiting, 1991) studies per- formed in the guinea pig spinal cord. The second objec- tive was to characterize the class of BK receptor pre- sents in the spinal cord using a series of highly selective agonists and antagonists. A preliminary report of this work has been presented (Lopes et al., 1992a).

MATERIALS AND METHODS Tissue preparation

Male Hartley guinea pigs, used according to the stan- dards of the Canadian Council for Animal Care and weighing 450-500 g (Charles River Lab., St-Constant, Quebec, Canada) (n = 8) were sacrificed by stunning and exsanguination and subjected to a dorsal laminec- tomy. In all cases, a longitudinal incision of the dura was made with care to avoid damage of the spinal cord and the appropriate cervical (C3-C8), thoracic (T3-T8), lumbar (L2-L,), and sacral (SlS4) segments of the spi- nal cord were removed and immediately frozen in 2-me- thylbutane at -40°C. A group of 8-10 pieces of spinal cord from cervical, thoracic, lumbar, and sacral seg-

ments were separately mounted in an embedding me- dium to form a block. Serial 20-pm coronal sections were cut on a cryostat (- 13"C), thaw-mounted on gela- tin-coated slides, desiccated overnight a t 4°C and kept at -80°C until used.

Iodination of the radioligand [Tyr'l-BK was iodinated and purified as described

previously (Tousignant et al., 1991). Briefly, 10 pg of [Tyr'l-BK, 1 mCi Na-lZ5I and 10 pg of Iodogen were incubated for 30 min at room temperature in 60 pl of borate buffer (50 mM, pH 8.5). [1251-Tyr81-BK was puri- fied using a C,, pBondapak column followed by a re- verse phase HPLC. A mixture of 1:l ammonium acetate and methanol (pH 7.5) was then used for 10 min, fol- lowed by a gradient of 50-80% methanol. The monoiodi- nated compound (specific activity of 500 Ci/mmol) was subsequently dissolved in water containing 1 mg/ml of bovine serum albumin (BSA) and frozen a t -60°C in small aliquots until used (2-3 days).

Peptides, drugs, and materials Radiolabelled Na-[lxI1, tritium microscales and 3H-

Hyperfilm were purchased from Amersham Canada (Oakville, Ontario, Canada). The C,, pBondapak col- umn was obtained from Waters Canada (Mississauga, Ontario, Canada). Piperazine-N,N'-bid2-ethanesulfo- nic acid1 (PIPES), 1,lO-phenanthroline, dithiothreitol, bacitracin, captopril, and bovine serum albumin were purchased from Sigma Chemical Co. (St. Louis, MO). BK was purchased from Hukabel Scientific Ltd. (Mont- real, Quebec, Canada), while [Tyr'l-BK (Anal) was ob- tained from IAF Biochem Canada (Montreal, Quebec, Canada). D-Ar~Hyp3,D-Phe7,Leu81-BK (ha,), D-Arg [Tyr3,D-Phe7,Leus]-BK (ha,), Tyro,D-Arg[Hyp3D-Phe7, Leua]-BK (Ana,), Tyr0,des-Arg1'-KD (Ana,), [Leua]-des- Argg-BK (Ana,), D-AI-&H~~~,T~~~~'D-P~~~I-BK (ha,), D-Arg[Hyp3,Leusl-BK (Ana,), D-Arg[Hyp3,Gly6,Leus]- BK ( h a l o ) were prepared using the solid-phase method and purified by high-pressure chromatography (Drapeau and Regoli, 1988). D-Arg[Hyp3,Thi5,D-Tic7,0ical-BK (Hoe 140; Ana,) was a generous gift of Hoechst AG (Frankfurt, Germany). All other chemicals were ob- tained from standard commercial sources. The selectiv- ity and antagonist activity of these compounds have been published elsewhere (Rhaleb et al., 1991; Regoli et al., 1992a,b).

In vitro receptor autoradiography For receptor autoradiography, sections were warmed

to room temperature and dried before use. Incubations were conducted for 90 min at room temperature using 280 pM [1251-Tyr81-BK in a medium containing 25 mM PIPES-NH40H buffer (pH 6.8, 4"C), 1 mM 1 , l O - phenanthroline, 1 mM dithiothreitol, 0.014% bacitra- cin, 0.1 mM captopril, 0.2% bovine serum albumin (pro- tease free) and 7.5 mM magnesium chloride. The non- specific binding was assessed in the presence of 1 pM

P. LOPES ET AL. 50

unlabelled [Tyr8]-BK. At the end of the incubation pe- riod, slides were transferred sequentially through four rinses of 4.5 min each in PIPES-NH,OH buffer at 4°C and rapidly dipped into cold distilled water to remove excess salts. Sections were then air-dried and juxta- posed against 3H-Hyperfilm for 10-12 days, along with tritiated microscales (Kar and Quirion, 1992; Yashpal et al., 1991). In order to characterize the BK receptor subtype, competition experiments were carried out un- der the same assay conditions, using several selective agonists and antagonists for the B, or B, receptors. These analogues were used in concentrations ranging from 0.1 pM to 1 pM. All competition binding assays were performed in duplicate using multiple sections at each time. The autoradiograms were quantified densit- ometrically using MCID image analysis system (Imag- ing Research Inc., Ontario, Canada). The specific bind- ing was determined by subtracting the non-specific binding (in the presence of 1 pM [Tyr'l-BK) from the total binding in each lamina from adjacent sections of cervical, thoracic, lumbar and sacral segments of the spinal cord (Molander et al., 1984; 1989). Under these assay conditions, specific [1251-Tyr81-BK binding repre- sented up to 90% of the totally bound ligand in the substantia gelatinosa.

For emulsion receptor autoradiography, a batch of sections were fixed by immersion in a solution 50 mM phosphate buffer (pH 7.4) containing 5% glutaralde- hyde and dehydrated in increasing concentrations of ethanol (70-100%, 5 midbath) (Hamel and Beaudet, 1984). Sections were then coated by dipping in Kodak NTB-2 emulsion diluted 1:l with distilled water and exposed for one month in light-tight boxes at 4°C (Gouarderes et al., 1991). Sections were then developed in freshly prepared D-19 (Kodak developer) for 5 min at 18"C, fixed in Kodak Ektaflo, stained in cresyl violet and coverslipped with permount before microscopic ex- amination.

Statistical analysis of data The results are expressed as means & SEM. Statisti-

cal significance of differences were evaluated with Stu- dent's t-test for unpaired samples. A one-way analysis of variance (ANOVA) was used for multiple compari- sons. Only probablity values (PI <0.05 were considered statistically significant.

RESULTS Autoradiographic localization of BK receptor

binding sites Film autoradiograms of [1251-Tyr8]-BK binding sites

are shown in Figure 1. Throughout all major levels of the guinea pig spinal cord, the overall distribution of [1251-Tyr8]-BK binding sites shows specific enrichments in the grey matter. In all cervical, thoracic, lumbar, and sacral segments studied, [1251-Tyr8]-BK binding sites are localized primarily to the superficial layers of the

dorsal horn (i.e., laminae I to 111). The substantia gela- tinosa (lamina 11) was shown to possess the highest density of binding sites (P < 0.001) in all segments (Figs. 1-3). A somewhat lower but still significant den- sity of [1251-Tyr8]-BK binding sites was noticed in lami- nae I and 111. Adjacent to the superficial layers of the dorsal horn, the Lissauer's tract showed a high density of BK binding sites (Fig. 1). Lower densities of [1251- Tyr'I-BK binding sites were found in deeper dorsal horn (laminae IV-V), ventral horn and intermediolat- era1 cell column (laminae VI-IX) and in the area sur- rounding the central canal (lamina X) (P < 0.001 in all segments). White matter exhibited a very low density of specific [1251-Tyr81-BK binding (Fig. 1). A rostrocaudal gradient of specific [1251-Tyr81-BK binding was also ob- served in superficial layers with values ranging from 2.2 fmol/mg tissue in lamina I in the cervical segment to 5.1 fmol/mg tissue in this lamina in the sacral segment (F(3,36) = 25.53; P < 0.001), and from 5.8 fmol/mg tis- sue in lamina I1 at the cervical level to 10.1 fmol/mg tissue in the sacral segment (F(3,36) = 20.91; P < 0.001). In laminae VII-X, the lumbar segments showed the highest amounts of specific ['251-Tyr8]-BK labelling (Fig. 3). The dark-field image (Fig. 1-j) reveals a lower density of grains in the grey matter than in the white matter in the presence of 1 pM unlabelled [Tyr'l- BK, suggesting a stronger nonspecific binding in the white matter.

At the cellular level, [1251-Tyr81-BK binding showed a similar profile, i.e., receptor binding sites being concen- trated in lamina I1 of the dorsal horn, while other lami- nae exhibited modest to low densities of specific label- ling (Fig. 2). At higher magnification, labelling was detected generally distributed over the neuropil with some neuronal perikarya showing a dense concentra- tion of specific binding sites. In the ventral horn, [1251- Tyr'I-BK binding was evenly scattered over the neuro- pi1 and perikarya of motoneurons, although some discrete silver grain alignments were apparent around the membrane of motoneuron perikarya and proximal processes (Fig. 2).

Competition of specific [1251-Tyral-BK binding Competition experiments using 280 pM [1251-Tyr81-

BK and 0.1 pM to 1 pM of the unlabelled BK related peptides revealed that [Tyr'l-BK and its analogues in- duce a concentration-dependent inhibition of [1251- Tyr'I-BK binding in the substantia gelatinosa of the

~~ ~

Fig. 1. Distribution of total [1251-Tyr81-BK binding sites in the guinea pig spinal cord. Autoradiograms of cervical (a), thoracic (c), lumbar (e), and sacral (g) transversal sections are shown along with their respective dark-field photomicrograph (b,d,f,h). Total binding is shown (a-h). Nonspecific binding is represented in sections i and j in the presence of 1 )IM [Tyr'l-BK. Note the high level of specific binding in the substantia gelatinosa (SG) and the Lissauer tract (LT). Other abbreviations are CC, central canal; DH, dorsal horn; VH, ventral horn; WM, white matter.

52 P. LOPES ET AL.

Fig. 2. Cellular localization of [1251-TyrSl-BK binding sites in the guinea pig spinal cord. Silver grains are detected throughout the sub- stantia gelatinosa, marginal layer and dorsal root entry zone (a: x125). Labelling is apparent over both the perikarya and neuropil ( b x 1251, with some perikarya showing an intense labelling (c: X400).

l 2 1

" I 11 111 IV v v1 VII VIIl IX x Laminae

Fig. 3. Quantification of specific ['251-TyrSl-BK binding sites in all laminae of cervical (01, thoracic (a], lumbar Caj, and sacral (m) seg- ments of the guinea pig spinal cord. Values represent the mean i S E M of 10 sections quantified in a random order from a group of 8 animals. The differences between lamina I1 and laminae I and I11 are statisti- cally significant (P c: 0.001) a t each segment of the spinal cord. The densities of specific binding were also significantly higher in laminae I and 111 when compared to laminae IV to X in cervical (F(1,87) = 190.90; P < 0.001), thoracic (F(1,87) = 207.14; P < 0.001), lumbar (F(1,84) = 21.312; P < 0.001) and sacral (F(1,88) = 145.48 P < 0.001) segments.

guinea pig spinal cord (Table I; Figs. 4, 5). The rank order of potency to inhibit [ 1251-Tyr8]-BK binding was: h a , (Hoe 140) ;>>> h a , >> h a , > h a , = h a , , >

Moderate to low labelling densities are detected in the ventral horn (d: x200), although some silver grain alignments were visible around motoneuron cellular membrane (short arrows) and along proximal processes (long arrows).

h a , 3 h a , > Ana, >>> h a , = Ana,. h a , (Hoe 140), the newly developed B, receptor antagonist (Wirth et al., 1991; Hock et al., 1991; Rhaleb et al., 19921, showed the highest potency with a K, value of 0.04 nM and a Hill coefficient (nH) of 2.90. The very high potency of this peptide to compete with [1251-Tyr8]- BK binding is illustrated by the fact that it competed 80-90% of the totally bound ligand at 50-100 pM (Fig. 5). [Tyr81-BK, the noniodinated peptide (Anal), inhib- ited the binding with one-tenth the potency of Hoe 140 (Ana,; K, = 0.6 nM) and a Hill coefficient value of 0.76 (Table 1). BK antagonists ( h a , , h a , ) with D-phenyl- alanine (D-Phe) residue in position 7, hydroxyproline (Hyp) or tyrosine (Tyr) in position 3, leucine (Leu) re- placing phenylalanine in position 8, and D-arginine (D-Arg) at the N-terminal showed very similar competi- tion curves as [Tyr'l-BK, although they were 20-56 times less potent (Table I). The addition of a Tyr and D - A r g residues at the N-terminal ( h a , ) or the replace- ment of the serine in position 6 by a glycine residue (Anal,) similarly decreased the potencies of these pep- tides to inhibit [ 1251-Tyr8]-BK binding. B2* ( h a , , h a , and Ana,,) and BZB ( h a , ) selective antagonists dem- onstrated similar potencies (Table I). In contrast, the selective B, agonist ( h a , ) and antagonist ( h a , ) did not inhibit [ 1251-Tyrs]-BK binding at pM concentration (Table I; Figs. 4,5).

BRADYKININ RECEPTORS IN THE GUINEA PIG SPINAL CORD

TABLE I . Primary structure, receptor selectiuity, inhibition constant (KJ and Hill coefficient (nH) ualues of BK analogues on [1251-TyrR1-BK binding in the substantia gelatinosa of the guinea pig spinal cord'

53

BK Y analogue Primary structure Receptor' (nM) n1< h a 1 Arg-Pro-Pro-Gly-Phe-Ser-Pro-Tyr-Arg-OH B2 0.6 0.76 h a 2 D-Arg-Arg-Pro-Hyp-Gly-Phe- Ser-D-Phe-Leu-Arg-OH BZA 12.4 0.74 h a 3 D-Arg-Arg-Pro-Tyr-Gly-Phe-Ser-D-Phe-Leu-Arg-OH B2B 34.5 0.96 h a 4 D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg-OH B2Ar BZB 0.04 2.90

Tyr-D-Arg-Arg-Pro-Hyp-Gly-Phe-Ser-D-Phe-Leu-Arg-OH BZA, B'B 23.4 1.03 - h a 5

>1,000 h a , Tyr-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-OH B, h a 7 H-Arg-Pro-Pro-GI y-Phe-Ser-Pro-Leu-OH Bl >1,000 h a , D-Arg-Arg-Hyp-Pro-Gly-Thi-Ser-D-Phe-Thi-Arg-OH B2A, B2B 43.5 0.99

-

h a , D-Arg-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Leu-Arg-OH BZA 33.5 1.14 h a l o D-Arg-Arg-Pro-Hyp-Gly-Phe-Gly-Pro-Leu-Arg-OH B2A 23.0 0.73

'Numbering system for amino acids is based on the BK sequence (Arg'-Pro2-Pro3-Gly4-Phe5-SerB-Pro7-Phes-~~-OH) 'Receptor selectivity according to Regoli and Barabe, 1980; Regoli et al., 1992a; 1992b. Definition: K, = IC,o(l + F/Kd).

10- l3 M

Anal

10- lo M M M

Ana,

AnaG

Ana9

Fig. 4. Concentration-dependent inhibition of specific l'2'I-Tyral-BK binding sites in the substantia gelatinosa of the lumbar segments of the guinea pig spinal cord by increasing concentrations (lo-'$- lO-,M) of various kinin analogues.

DISCUSSION Using in vitro receptor autoradiography, the present

study demonstrates the detailed anatomical localiza- tion of specific [1251-Tyr8]-BK binding sites a t all major segmental levels of the guinea pig spinal cord. These receptors were quantified in various laminae through- out the spinal cord and pharmacologically character- ized as being of the B, type. The presence of high densi-

ties of binding sites in superficial laminae suggests a role for BKin the modulation or transmission of nocicep- tive information.

Distribution of ['251-TyrS]-BK binding sites [ 1251-Tyr8]-BK binding displays a differential pattern

of distribution in the guinea pig spina€ cord. The high- est density of specific [1251-Tyr8]-BK binding sites are

54 P. LOPES ET AL.

r- , -13 -12 -11 -10 -9 -8 -7 -6

LOG [analogue], M

Fig. 5. L1"5I-TyrRj-BK competition binding curves in the presence of h a , (m), h a , (+ ), h a , (A), h a , (o), and h a , {*) in the substantia gelatinosa of guinea pig lumbar spinal cord. Ordinate: concentration expressed as a percentage of the total [lZ5I-TyrRJ-BK binding in the presence of 0.1 pM of unlabelled tTyr'JI-BK. Abscissa: log of molar concentration of the analogue.

located in superficial layers of the dorsal horn, particu- larly in the substantia gelatinosa and lamina I, whereas other laminae exhibited moderate to low den- sities of specific labelling. Through the use of high- resolution emulsion autoradiography, the localization of [1251-Tyr81-BK binding sites was confirmed to be mostly confined to the substantia gelatinosa, with some perikarya showing a high density of specific labelling. This pattern of distribution is in accordance with quali- tative reports on the localization of spinal BK receptor sites (Steranka et al., 1988; Fujiwara et al., 1989; Sharif and Whiting, 1991) and provides additional in- formation on the quantity of specific sites present in various segments of the guinea pig spinal cord. In the rat, a similar distribution of [1251-Tyrsl-BK has been demonstrated, although quantitative values were lower than those presently observed in the guinea pig spinal cord (Lopes et al., 1992b).

Similarly to BK receptors, other peptide-binding sites possess a differential pattern of distribution in the grey matter of the spinal cord. For example, neuroki- nins (Helke et al., 1986; Yasphal et al., 19901, vasoac- tive intestinal polypeptide (VIP) (Yashpal et al., 19911, neuropeptide Y (NPY) (Kar and Quirion, 1992), galanin (Arvidsson et al., 1991; Melander et al., 1988) and opi- oid (LaMotte et al., 1976; Gouarderes et al., 1991; Besse et al, 1992) receptors are also preferentially concen- trated in superficial laminae of dorsal horn. This fur- ther reveals the complexity of the chemical neuroanat- omy of the substantia gelatinosa, at least in regard to peptidergic innervation.

Our data also demonstate the presence of a rostro- caudal gradient of [ 1251-TyrS]-BK binding within the substantia gelatinosa; the sacral segment being partic- ularly enriched. Although the physiological signifi- cance of this gradient remains to be clearly established, it correlates well with similar observations reported for other peptide receptors including neurokinins (Vita et al., 1990; Yashpal et al., 1990), VIP (Yashpal et al.,

1991) and kappa opioid binding sites (Besse et al., 1991; Gouarderes et al., 1985). It would thus be of interest to determine if BK receptor gradient relates to the density of receptors per nerve terminal as reported for CGRP and opioids (Gouarderes et al., 1991; Yashpal et al., 1992; Garry et al., 1991).

It is known that certain peptide receptor binding sites are localized presynaptically to sensory nerve ter- minals (opioids) (Besse et al., 1990; LaMotte et al., 1976; Gouarderes et al., 1991) while others are postsyn- aptic (SP and CGRP) (Helke 1986; Yashpal et al., 1992; Garry et al., 1991) in the superficial laminae of the dorsal horn. As for BK binding sites, results from ear- lier studies (Steranka et al., 1988, Lopes et al., 1992b) suggest a presynaptic localization. For example, in the rat, we have shown that the section of the sciatic nerve or a lumbar unilateral rhizotomy greatly decreased [1251-Tyrsl-BK binding in the lesioned side (Lopes et al., 1992b). Moreover, the finding that a neonatal treat- ment with capsaicin, a toxin affecting preferentially unmyelinated primary afferent C-fibers (Fitzgerald 1983) induced an important loss in [1251-Tyrsl-BK bind- ing in lamina I1 (Lopes et al., 1992b) also supports a presynaptic localization. Additionally, r3H1BK binding sites are present in a subset of cells in the dorsal root ganglia (Steranka et al., 1988), bringing further evi- dence that BK binding sites may possibly be associated with fine diameter primary afferent fibers terminating in laminae I and I1 of the spinal cord. Nonetheless, a postsynaptic localization can not be fully excluded since a population of BK receptors appears to be involved in the modulation of noradrenergic bulbospinal fibers (La- neuville et al., 1989) as well as preganglionic sympa- thetic neurones (Lopes and Couture, 1992).

In the intermediolateral column and the ventral horn, specific BK labelling detected using high resolu- tion emulsion autoradiography was scattered through- out the neuropil suggesting that [1251-Tyr81-BK binding sites are not necessarily associated with any peculiar cell type. However, the presence of silver grain align- ments along the perikarya and proximal dendrites of certain motoneurons suggest the existence of BK recep- tors in close apposition to these cells. A similar distribu- tion was previously reported for mu opioid binding sites on ventral horn motoneurons (Gouarderes et al., 1991 ). It is also likely that a certain proportion of specific BK binding sites are located either on interneurons, on de- scending projecting fibres, and/or on small population of large diameter sensory fibers terminating in the ven- tral horn. These sites could thus account, as proposed for opioids (Gouarderes et al., 1991; Besse et al., 19921, for the direct effect of BK on the integration of sympa- thetic autonomic functions at the spinal level.

Pharmacological characterization of specific [1251-Tyr8]-BK binding sites

A comparison of the relative potencies of various ana- logues in competing for specific [1251-Tyrsl-BK binding

BRADYKININ RECEPTORS IN THE GUINEA PIG SPINAL CORD 55

sites allowed the classification of these compounds into three groups. h a , and h a , (Hoe 1401 displayed the highest affinities; Hoe 140 showing a 10-fold greater potency for this site than the unlabelled [Tyr'l-BK and a 1,000 times higher affinity than h a , . This is consis- tent with previous reports that have shown that ~-Arg[Hyp~,Thi~,~-Tic~,Tic~l-BK and Hoe 140 were two to three orders of magnitude more potent than Ana, in the guinea pig ileum (Farmer et al., 1991; Seguin et al., 1992). Furthermore, the finding that Hoe 140 has a Hill coefficient (n, = 2.90) much higher than those of all the other analogues suggests that this analogue may bind in a nonequilibrium or noncompetitive manner (Lim- bird, 1986). This would also support early observations indicating that Hoe 140 occupies the BK receptor for a prolonged period of time both in vivo and in vitro (Wirth et al., 1991; Hock et al., 1991; Lembeck et al., 1991). A similar value (n, = 2.36) was also reported on the iso- lated rabbit jugular vein (Regoli et al., 1992a; Rhaleb et al., 1991), contrasting with the much lower Hill coeffi- cient (n, = 0.96) found in guinea pig tracheal mem- brane preparations (Wirth et al., 1991).

These apparent discrepancies could be explained by a recent study which reported the existence of at least three subtypes of BK binding sites, namely, the BU, BZB, and B,, types (Seguin et al., 1992). These authors reported that in both brain and lung preparations, Hoe 140 ( h a , ) was able to discriminate between two sites with a 1000-fold difference in affinity. The B,, subtype, which constituted 80% of total [1251-Tyr81-BK binding, was classified as the high-affinity site, whereas the BZc represented the low-affinity binding component. It would be premature to speculate, based only on & val- ues, that BK binding sites present in the guinea pig spinal cord are of the B,, subtype. However, it is of interest to note that the affinity of Hoe 140 (Ana,) found in our assay is closer to the K, values described for the high-affinity binding component present in the guinea pig lung and in the brain.

A second group of analogues consisting of Ana,, Ana,, Ana,, Ana,, h a , and Analo, competes for [1251-Tyr81- BK binding sites with affinities in the 10 to 50 nM range. The combination of Hyp3, ~-Phe,, and Leus thus seems to be essential for a strong antagonist activity on the B,, receptor subtype as Ana, was shown to be 100 times more potent in a BZA-containing tissue (rabbit jugular vein) than in a B2B preparation (guinea pig ileum) (Rhaleb et al., 1992; Regoli et al., 1992a). In guinea pig ileum epithelial membranes, Ana, was also reported to possess an affinity markedly lower than that of h a , in competing for [lz5I-Tyr81-BK binding (Tousignant et al., 1992). Likewise, deletion of ~ - P h e ~ greatly diminished the antagonist activity of Ana, and h a l o for the B,B receptor making them appropriate tools to discriminate between the BU and BzB receptor subtypes. Replacement of Hyp3 in Ana, by a tyrosine residue produced a pure antagonist ( h a , ) on the B2B sites and a partial to full agonist in BZA tissues such as

the rabbit jugular vein, the vena cava and the urinary bladder (Regoli et al., 1992a). However, in the guinea pig spinal cord (present study) and the guinea pig ileum epithelial membranes (Tousignant et al., 19921, the ad- dition of a tyrosine residue to the N-terminal (Ana,) did not modify their affinities for [1251-Tyrsl-BK sites. Thus, a clear distinction between the B,, and the B2B sub- types could not be achieved in our study; further exper- iments await the development of more discriminating tools.

The third group, represented by the selective B, ago- nist (ha,) and antagonist (ha , ) , was unable to com- pete for [1251-Tyr81-BK binding sites at concentrations up to 1.0 pM. This demonstrates that BK binding sites present in the guinea pig spinal cord are unlikely to be of the B, subtype. This is consistent with earlier studies (Fujiwara et al., 1989; Privitera et al., 1991; Sharif and Whiting, 1991).

The main findings obtained in the present study with the BK analogues are in agreement with a functional study performed in the conscious rat (Lopes et al., 1993). In the latter study, we found that the cardiovas- cular responses to intrathecal BK were unaffected by the prior injection of a B, receptor antagonist (Ana,) while they were dose-dependently and reversibly atten- uated by four B, antagonists (Ana,, Ana,, Ana, (Hoe 140) and ha,) . In addition, the B, receptor agonist des-Argg-BK was inactive in this paradigm and on ther- monociception (Laneuville and Couture, 1987).

Functional significance Most physiological studies on the role of kinins in the

spinal cord relate to the modulation of nociceptive infor- mation. For example, BK, upon its application to the spinal cord was shown to activate nociceptive dorsal horn neurones (Dunn and Rang, 1990; Baccaglini and Hogan, 1983), to cause behavioral changes and to alter nociceptive reflex activity (Laneuville and Couture, 1987; Lopes and Couture, 1992). The elevated kinin production following experimental spinal cord injury (Xu et al., 1991), and the presence of [,HI-BK binding sites in the dorsal root ganglia (Steranka et al., 1988) also supports a role for BK in this mechanism. Thus, the present data demonstrating a high density of [lZ5I- Tyr'l-BK binding sites in the superficial layers of the dorsal horn are consistent with an implication of BK, along with several other peptides, in the processing of nociceptive information.

The dorsal horn of the spinal cord, particularly the substantia gelatinosa and lamina I are the major sites of termination of thin sensory myelinated (A&) and non- myelinated (C) fibers carrying nociceptive and non-no- ciceptive informations (Salt and Hill, 1983; Willis and Coggeshall, 1991). The selective destruction of these fibers with the neurotoxin capsaicin or by the mechani- cal interruption of all inputs (rhizotomy or sciatic nerve section) induced the marked depletion of various pep- tides receptors, including opioids (Gouarderes et al.,

56 P. LOPES ET AL

1991) and ['251-"yr8]-BK (Lopes et al., 199213) from lam- inae I and 11, suggesting their presynaptic localization. By acting presynaptically on afferent terminals, BK has recently been proposed to induce the release of SP and CGRP (Andreeva and Rang, 1993; Schaible et al., 1992), this in turn could promote hyperalgesia. Al- though the intracellular mechanisms by which BK may induce the release of these peptides remains to be es- tablished, such an action is consistent with the nocicep- tive responses elicited by the intrathecal administra- tion of BK-related peptides in rats (Laneuville and Couture, 1987; Lopes and Couture, 1992). Much evi- dence for the presence of dense immunoreactive net- work of SP, CGRP, and opioids in the substantia gelati- nosa has been reported (Klein et al., 1991; Yashpal et al., 1992, Pohl et al., 1990), correlating with the exist- ence of their respective binding sites. However, the lack of information concerning the precise site of synthesis and storage of BK in the spinal cord hinders progress regarding the physiological relevance for this peptide in this tissue. In this context, the development of appro- priate tools, such as highly specific BK antibodies, are needed for the confirmation of a role for kinins in noci- ceptive pathways and to provide anatomical support for the presence of BK-like peptides in various cellular ele- ments in the spinal cord.

Interestingly, a rather low but significant density of specific ['251-Tyr8]-BK binding sites was also detected in the intermediolateral horn where cell bodies and dendrites of preganglionic sympathetic fibers are lo- cated. These specific BK sites may mediate the spinal action of kinins on the cardiovascular system, although one cannot exclude the possibility that kinins activates preganglionic sympathetic fibers through interneurons and others transmitters present in the area.

In summary, it appears that specific BK binding sites found in the spinal cord represent functional BK recep- tors. Furthermore, the present study also supports the hypothesis that BK may play a role in the modulation of nociceptive information, as BK receptors appear to be mostly localized to areas densily innervated by small diameter sensory fibers. The finding that the highly potent B, receptor antagonist (Hoe 140; Ana,) inhibited [1251-Tyr81-BK binding with an affinity in the low nano- molar range, while the B, receptor antagonist ([Leus]- des-Argg-BK) and agonist (Tyr0,des-Arg1'-KD) were in- active, indicates that the guinea pig spinal cord is enriched with B, receptors; the exact subtype (B2*, B2B, and Bzc) remaining to be fully established.

ACKNOWLEDGMENTS This work was supported by grants in aid from the

Medical Research Council of Canada (MRCC) to R.C. and R.Q. R.Q. and R.C. are senior scholars of the Fonds de la Recherche en Sante du Quebec (FRSQ). S.K. is a postdoctoral fellow of the FRSQ, D.R. is a career inves- tigator of the MRCC, and P.L. holds studentships from

the Heart and Stroke Foundation of Canada and the Fonds pour la Formation de Chercheurs et l'Aide a la Recherche. We thank Daniella Jukic for the synthesis of the kinin-related peptides used in this study. The technical assistance of Jeanne Lavoie and Feliciana Faraco Cantin and the graphic work of Claude Gauth- ier and Daniel Cyr are gratefully acknowledged.

REFERENCES Andreeva, L., and Rang, H.P. (1993) Effect of bradykinin and pros-

taglandins on the release of calcitonin gene-related peptide-like im- munoreactivity from the spinal cord in vitro. Br. J . Pharmacol., 108:185-190.

Arvidsson, U., Ulfhake, B., Cullheim, S., Bergstrand, A,, Theodorsson, E., and Hokfelt, T. (1991) Distribution of "'I-galanin binding sites, immunoreactive galanin, and its coexistence with 5-hydroxy- tryptamine in the cat spinal cord: biochemical, histochemical, and experimental studies at the light and electron microscopic level. J. Comp. Neurol., 308:115-138.

Baccaglini, P.I., and Hogan, P.G. (1983) Some rat sensory neurones in culture express characteristics of differentiated pain sensory cells. Proc. Natl. Acad. Sci. USA, 803596598.

Bathon, J.M. and Proud, D. (1991) Bradykinin antagonists. Annu. Rev. Pharmacol. Toxicol., 31:129-162.

Besse, D., Lombard, M.C., Zajac, J.M., Roques, B.P., and Besson, J.M. (1990) Pre- and postsynaptic distribution of mu, delta and kappa opioid receptors in the superficial layers of the cervical dorsal horn of the rat spinal cord. Brain Res., 521:15-22.

Besse, D., Lombard, M.C., and Besson, J.M. (1991) Autoradiographic distribution of mu, delta and kappa opioid binding sites in the super- ficial dorsal horn, over the rostrocaudal axis of the rat spinal cord. Brain Res., 548:287-291.

Besse, D., Lombard, M.C., Perrot, S., and Besson, J.M. (1992) Regula- tion of opioid binding sites in the superficial dorsal horn of the rat spinal cord following loose ligation of the sciatic nerve: Comparison with sciatic nerve section and lumbar dorsal rhizotomy. Neuro- science, 50:921-933.

Bhoola, K.D., Fimeroa, C.D., and Worthv, K. (1992) Bioremlation of kinins: kallikreins, kininogens, and kininases. Pharrn&ol. Rev.,

Braas, K.M., Manning, D.C., Perry, D.C., and Snyder, S.H. (1988) Bradykinin analogues: Differential agonist and antagonist activi- ties suggesting multiple receptors. Br. J. Pharmacol., 94:3-5.

Burch, R.M., and Kyle, D.J. (1992) Mini Review, Recent developments in the understanding of bradykinin receptors. Life Sci. 50:829-838.

Drapeau, G., and Regoli, D. (1988) Synthesis of bradykinin analogs. In: Immunochemical techniques: Part n. Chemotaxis and Inflam- mation. Methods Enzymol., 163:263-272.

Dunn, P.M. and Rang, H.P. (1990) Bradykinin-induced depolarization of primary afferent nerve terminals in the neonatal rat spinal cord in vitro. Br. J. Pharmacol., 100:65&460.

Farmer, S.G., Burch, R.M., Kyle, D.J., Martin, J.A. Meeker, S.N., and Togo, J. (1991) D-Arg[Hyp3,Thi5,D-Tic7,Ticsl-bradykinin, a potent antagonist of smooth muscle BK, receptors and BK, receptors. Br. J. Pharmacol., 102:785-787.

Fitzgerald, M. (1983) Capsaicin and sensory neurones: A review, Pain,

Fujiwara, Y., Mantione, C.R., Vavrek, R.J., Stewart, J.M., and Yama- mura, H.I. (1989) Characterization of c3H1bradykinin binding sites in guinea-pig central nervous system: possible existence of B, sub- types. Life Sci. 44:1645-1653.

Garry, M.G., Kajander, K.C., Bennet, G.J., and Seybold, V.S. (1991) Quantitative autoradiographic analysis of 1'2511-human CGRP bind- ing sites in the dorsal horn of rat following chronic constriction injury or dorsal rhizotomy. Peptides, 12:1365-1373.

Gouarderes, C., Beaudet, A., Zajac, J.-M., Cros, J . , and Quirion, R. (1991) High resolution radioautographic localization of ['2511FK-33- 824-labelled mu opioid receptors in the spinal cord of normal and deafferented rats. Neuroscience, 43:197-209.

Gouarderes, C., Cros., J . , and Quirion, R. (1985) Autoradiographic localization of mu, delta and kappa opioid receptor binding sites in rat and guinea pig spinal cord. Neuropeptides, 6:331342.

Hamel, E., and Beaudet, A. (1984) Localization of opioid binding sites in rat brain by electron microscopic radioautography. J. Elect. Mi- crosc. Tech., 1:317-329.

Helke, C.J., Charlton, C.G., and Wiley, R.G. (1986) Studies on the cellular localization of spinal cord substance P receptors. Neuro- science, 19523-533,

44:l-80.

15:109-130.

BRADYKININ RECEPTORS IN THE GUINEA PIG SPINAL CORD 57

Henry, J.L. (1976) Effects of substance P on functionally identified units in cat spinal cord. Brain Res., 114:439451.

Hock, F.J., Wirth, K., Albus, U., Linz, W., Gerhards, H.J., Wiemer, G., Henke, St., Breipohl, G., Konig, W., Knolle, J., and Scholkens, B.A. (1991) Hoe 140 a new potent and long acting bradykinin-antagonist: in vitro studies. Br. J . Pharmacol., 102:769-773.

Kar, S. and Quirion, R. (1992) Quantitative autoradiographic localiza- tion of ['2sI]neuropeptide Y receptor binding sites in rat spinal cord and the effects of neonatal capsaicin, dorsal rhizotomy and periph- eral axotomy. Brain Res., 574:333-337.

Klein, C.M., Guillamondegui, O., Krenek, C.D., La Forte, R.A., and Coggeshall, R.E. (1991) Do neuropeptides in the dorsal horn change if the dorsal root ganglion cell death that normally accompanies peripheral nerve transection is prevented? Brain Res., 552:273-282.

LaMotte, C.C., Pert, C.B., and Snyder, S.H. (1976) Opiate receptor binding in primate spinal cord: Distribution and changes after dor- sal root section. Brain Res., 112:407-412.

Laneuville, O., and Couture R. (1987) Bradykinin analogue blocks bradykinin-induced inhibition of a spinal nociceptive reflex in the rat. Eur. J. Pharmacol., 137:281-285.

Laneuville, O., Reader, T.A., and Couture, R. (1989) Intrathecal bradykinin acts presynaptically on spinal noradrenergic terminals to produce antinociception in the rat. Eur. J . Pharmacol., 159:273-283.

Lembeck, F., Griesbacher, T., Eckhardt, M., Henke, S., Breipohl, G., and Knolle, J . (1991) New, long-acting, potent bradykinin antago- nists. Br. J. Pharmacol., 102:297-304.

Limbird, L.E. (1986) Identification of neurotransmitter receptors us- ing radioligand binding techniques. In: Cell Surface Receptors: A Short Course on Theory and Methods. L.E. Limbird, ed. Martinus Nijhoff, Boston, pp. 1-194.

Lopes, P., and Couture, R. (1992) Cardiovascular responses elicited by intrathecal kinins in the conscious rat. Eur. J. Pharmacol., 210:137- 147.

Lopes, P., Kar, S., Tousignant, C., Regoli, D., Quirion, R. and Couture, R. (1992a) Autoradiographic localisation of '251-Tyrs-bradykinin binding sites in the guinea pig spinal cord. XIIth Washington Inter- national Spring Symposium on Growth Factors, Peptides and Re- ceptors, Washington, Abs. 84.

Lopes, P., Kar, S., Tousignant, C., Regoli, D., Quirion, R. and Couture, R. (1992b) Characterization of bradykinin receptors in the rat spinal cord. F.A.S.E.B. Abst., 1984.

Lopes, P., Regoli, D., and Couture, R. (1993) Cardiovascular effects of intrathecally administered bradykinin in the rat: Characterization of receptors with antagonists. Br. J . Pharmacol., in press.

Manning, D.C., Vavrek, R., Stewart, J.M., and Snyder, S.H. (1986) Two bradykinin binding sites with picomolar affinities. J . Pharma- col. Exp. Ther., '237504-512.

Marceau, F., Lussier, A. Regoli, D., and Giroud, J.P. (1983) Pharmacol- ogy of kinins: Their relevance to tissue injury and inflammation. Gen. Pharmacol., 14:209-229.

Melander, T., Kohler, C., Nilsson, S., Hokfelt, T., Brodin, E., Theodors- son, E., and Bartfai, T. (1988) Autoradiographic quantitation and anatomical mapping of '"I-galanin binding sites in the rat central nervous system. J. Chem. Neuoranat., 1:213-233.

Molander, C., Xu, Q., and Grant, G. (1984) The cytoarchitectonic orga- nization of the spinal cord in the rat. I. The lower thoracic and lumbosacral cord. J. Comp. Neurol., 230:133-141.

Molander, C., Xu, Q., Rivero-Melian, C., and Grant, G. (1989) Cytoar- chitectonic organization of the spinal cord in the rat. 11. The Cervical and upper thoracic cord. J. Comp. Neurol., 289:375-385.

Perry, D.C., and Snyder, S.H. (1984) Identification of bradykinin in mammalian brain. J. Neurochem., 43:1072-1080.

Pohl, M., Benoliel, J.J., Bourgoin, S., Lombard, M.C., Mauborgne, A,, Taquet, H., Carayon, A,, Besson, J.M., Cesselin, F., and Hamon, M. (1990) Regional distribution of calcitonin gene-related peptide-, sub- stance P-, cholescystokinin-, Met5-enkephalin-, and dynorphin A (1-81-like materials in the spinal cord and dorsal root ganglia of adult rats: effects of dorsal rhizotomy and neonatal capsaicin. J . Neurochem., 55: 1122-1 130.

Privitera, P.J., Daum, P.R., Hill, D.R., and Hiley, C.R. (1991) Autorad- iographic visualization and characteristics of ['2sIlbradykinin bind- ing sites in guinea pig brain. Brain Res., 577:73-79.

Rhaleb, N-E., Rouissi, N., Jukic, D., Regoli, D., Henke, S., Breipohl, G., and Knolle, J . (1992) Pharmacological characterization of a new highly potent B, receptor antagonist (HOE 140: ~-ArglHyp~,Thi',D- Tic7,0ic81bradykinin). Eur. J. Pharmacol., 210:115-120.

Rhaleb, N-E., Telemaque, S., Rouissi, N., Dion, S., Jukic, D., Drapeau, G., and Regoli, D. (1991) Structure-activity studies of bradykinin and related peptides. B,-receptor antagonists. Hypertension, 17:107-115.

Regoli, D., Jukic, D., Tousignant, C., and Rhaleb, N.-E. (1992a) Kinin receptor classification. Agent Actions, 38:475486.

Regoli, D., Rhaleb, N.-E., Dion, S., and Drapeau, G. (199213) New selective bradykinin receptor antagonists and bradykinin B, recep- tor characterization. TIPS, 11:156161.

Regoli, D., and Barabe, J . (1980) Pharmacology of bradykinin and related kinins. Pharmacol. Rev., 32:146.

Regoli, D. (1984) Neurohumoral regulation of precapillary vessels: The kallikrein-kinin system. J . Cardiovasc. Pharmacol., 6:S4014412.

Rifo, J., Pourrat, M., Vavrek, R.J., Stewart, J.M., and Huidobro-Toro, J.P. (1987) Bradykinin-receptor antagonists used to characterize the heterogeneity of bradykinin-induced responses in the rat vas deferens. Eur. J . Pharmacol., 142:305-312.

Saha, J.K., Sengupta, J.N., and Goyal, R.K. (1989) Effect ofbradykinin on opossum esophageal longitudinal smooth muscle: Evidence for novel bradykinin receptors. J. Pharmacol. Exp. Ther., 252:1012- 1020.

Salt, T.E., and Hill, R.G. (1983) Neurotransmitter candidates of soma- tosensory primary afferent fibres. Neuroscience, 10:1083-1103.

Schaible, H.-G., Hope, P.J., Lang, C.W., and Duggan, A.W. (1992) Calcitonin gene-related peptide causes intraspinal spreading of sub- stance P released by peripheral stimulation. Eur. J . Neurosci., 4:750-757.

Seguin, L., Widdowson, P.S., and Giesen-Crouse, E. (1992) Existence of three subtypes of bradykinin B, receptors in guinea pig. J. Neuro- chem., 59:2125-2133.

Sharif, N.A., and Whiting, R.L. (1991) Identification of B,-bradykinin receptors in guinea pig brain regions, spinal cord and peripheral tissues. Neurochem. Int., 18:89-96.

Steranka, L.R., Manning, D.C., DeHaas, C.J., Ferkany, J.W., Borosky, S.A., Connor, J.R., Vavrek, R.J., Stewart, J.M., and Snyder, S.H. (1988) Bradykinin as a pain mediator: receptors are localized to sensory neurons, and antagonists have analgesic actions. Proc. Natl. Acad. Sci. USA, 85:3245-3249.

Tousignant, C., Regoli, D., Rhaleb, N.-E., Jukic, D., and Guillemette, G. (1992) Characterization of a novel binding site for '2sI-Tyr-D-Arg- [Hyp3,D-Phe7,Leualbradykinin on epithelial membranes of guinea pig ileum. Eur. J . Pharmacol. (Mol. Pharmacol. Sec.), 225:235-244.

Tousignant, C., Guillemette, G., Barabe, J., Rhaleb, N.-E., and Regoli, D. (1991) Characterization of kinins binding sites: Identity of B, receptors in the epithelium and the smooth muscle of the guinea pig ileum. Can. J . Physiol. Pharmacol., 69:818-825.

Vita, G., Haunt, C.K., Hawkins, E.F., and Engel, W.K. (1990) Effects of experimental spinal cord transection on substance P receptors: A quantitative autoradiography study. Neuropeptides, 17:147-153.

Willis, W.D., and Coggeshall, R.E. (eds.), (1991) Sensory Mechanism of the Spinal Cord. 2nd Ed. Plenum Press, New York.

Wirth, K., Hock, F.J., Albus, U., Linz, W., Alpermann, H.G., Anag- nostopoulos, H., Henke, St., Breipohl, G., Konig, W., Knolle, J., and Scholkens, B.A. (1991) Hoe 140 a new potent and long acting brady- kinin-antagonist: in vivo studies. Br. J . Pharmacol., 102:774-777.

Xu, J., Hsu, C.Y., Junker, H., Chao, S., Hogan, E.L., and Chao, J. (1991) Kininogen and kinin in experimental spinal cord injury. J. Neurochem., 57:975-980.

Yashpal, K., Dam, T.V., and Quirion, R. (1990) Quantitative autora- diographic distribution of multiple neurokinin binding sites in rat spinal cord. Brain Res., 506:259-266.

Yashpal, K., Sarrieau, A,, and Quirion, R. (1991) ['2"IlVasoactive in- testinal polypeptide binding sites: Quantitative autoradiographic distribution in the rat spinal cord. J. Chem. Neuroanat., 4:439-446.

Yashpal, K., Kar, S., Dennis, T., and Quirion, R. (1992) Quantitative autoradiographic distribution of calcitonin gene-related peptide (hCGRPa) binding sites in the rat and monkey spinal cord. J . Comp. Neurol., 3223224-232.