distribution of bradykinin b2 receptors in sheep brain and spinal cord visualized by in vitro...

Distribution of Bradykinin B2 Receptorsin Sheep Brain and Spinal Cord Visualized

by In Vitro Autoradiography

C. MURONE,* G. PAXINOS,2 M.J. MCKINLEY,1 B.J. OLDFIELD,1 W. MULLER-ESTERL,3

F.A.O. MENDELSOHN,1 AND S.Y. CHAI1

1Howard Florey Institute of Experimental Physiology and Medicine,University of Melbourne, Parkville, 3052, Victoria, Australia

2School of Psychology, University of New South Wales, Kensington, 2033,New South Wales, Australia

3 Institute for Physiological Chemistry and Pathobiochemistry,Johannes-Gutenberg University at Mainz, Mainz, D-55099, Germany

ABSTRACTBradykinin B2 receptors were localized in the sheep brain and spinal cord by quantitative

in vitro autoradiography using a radiolabelled and specific bradykinin B2 receptor antagonistanalogue, 3-4-hydroxyphenyl-propionyl-D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin, (HPP-HOE140). This radioligand displays high affinity and specificity for bradykinin B2 receptors.The respective Ki values of 0.32, 1.37 and 156 nM were obtained for bradykinin, HOE140 andD-Arg[Hyp3,D-Phe7,Leu8]bradykinin competing for radioligand binding to lamina II of sheepspinal cord sections. Using this radioligand, we have demonstrated the distribution ofbradykinin B2 receptors in many brain regions which have not been previously reported.

The highest density of bradykinin B2 receptors occur in the pleoglial periaqueductal gray,oculomotor and trochlear nuclei and the circumventricular organs. Moderate densities ofreceptors occur in the substantia nigra, particularly the reticular part, the posterior thalamicand subthalamic nuclei, zona incerta, the red and pontine nuclei, some of the pretectal nucleiand in discrete layers of the superior colliculus. In the hindbrain, moderate levels ofbradykinin B2 receptor binding occur in the nucleus of the solitary tract, and in spinaltrigeminal, inferior olivary, cuneate and vestibular nuclei. Laminae II, X and dorsal rootganglia display the most striking binding densities in the spinal cord, while the remainder ofthe dorsal and ventral horn display a low and diffuse density of binding. Bradykinin B2receptors are extensively distributed throughout the sheep brain and spinal cord, not only tosensory areas but also to areas involved in motor activity. J. Comp. Neurol. 381:203–218,1997. r 1997 Wiley-Liss, Inc.

Indexing terms: localization; central nervous system; kinin; circumventricular organs

Bradykinin is a nonapeptide formed from the action ofthe enzyme kallikrein on its substrate kininogen. Thispeptide has been implicated in the contraction of smoothmuscle cells, oedema formation, inflammation, nocicep-tion, and cardiovascular homeostasis (Regoli and Barabe,1980; Marceau et al., 1983; Bhoola et al., 1992; Dray andPerkins, 1993). Bradykinin acts on at least two types ofreceptors, the B1 and the B2 receptors. The bradykinin B1receptor subtype is characterized by its high-affinity bind-ing to the agonists des-Arg9-bradykinin and des-Arg10-bradykinin and by the antagonists des-Arg9[Leu8]bradykininand des-Arg10Lys[Leu8]bradykinin (Bathon and Proud,1991). Generally, the bradykinin B1 receptor is not consti-tutively expressed in healthy tissue; instead, it is induced

by tissue damage or inflammation (Marceau et al., 1983;Bathon and Proud, 1991) and is limited in its distribution(Marceau, 1995). The bradykinin B2 receptor subtype hasbeen more extensively characterized and is considered tomediate the majority of the biological actions of bradyki-

Contract grant sponsors: National Health andMedical Research Council,the National Heart Foundation of Australia, the Austin Hospital MedicalResearch Foundation, the Deutsche Forschungsgemeinschaft.*Correspondence to: Carmelina Murone, Howard Florey Institute of

Experimental Physiology and Medicine, University of Melbourne, Park-ville, 3052, Victoria, Australia. E-mail: [email protected] 6 August 1996; Revised 25 November 1996; Accepted 22

December 1996

THE JOURNAL OF COMPARATIVE NEUROLOGY 381:203–218 (1997)

r 1997 WILEY-LISS, INC.

nin. Activation of the bradykinin B2 receptors on sensoryfibres induces pain in response to inflammation and facili-tates the release of noradrenaline from sympathetic nerveterminals (Regoli et al., 1990). Bradykinin B2 receptorsalso stimulate the release of nitric oxide and prostacyclinand increase intracellular calcium in cultured brain capil-lary endothelial cells (Doctrow et al., 1994; Wiemer et al.,1994), which may increase blood flow and vascular perme-ability.Over the past decade, components of the kallikrein-

kinin system have been reported in the central nervoussystem. Kininogenase activity and immunoreactive kal-likrein have been reported in the hypothalamus, cortex,pons, medulla, basal ganglia, and cerebellum of the ratbrain (Scicli et al., 1984). Similarly, tissue kallikrein-likeactivity and kallikrein mRNA is found in the pituitary andpineal glands and in the hypothalamus, cortex, cerebel-lum, and brainstem of rat brain (Chao et al., 1987).Central application of bradykinin elicits a variety of

effects, such as behavioural changes (Okada et al., 1977),

antidiuresis (Hoffman and Schmid, 1978), electroencepha-logram changes (Kariya and Yamauchi, 1981), and antino-ciception (Laneuville et al., 1989).Moreover, central admin-istration of bradykinin increases heart rate and bloodpressure (Diz, 1985; Lindsey et al., 1989; Privitera, 1992;Fior et al., 1993; Madeddu et al., 1994; Privitera et al.,1994) via bradykinin B2 receptors (Lindsey et al., 1989;Madeddu et al., 1990; Privitera, 1992; Privitera et al.,1994).Although much has been reported on the effects of

central administration of bradykinin, little is known aboutthe distribution of this peptide or its receptors. Immunore-active bradykinin-like activity was detected in the hypo-thalamus (Correa et al., 1979; Perry and Snyder, 1984),pituitary gland, medulla oblongata, cerebellum, and cortexof the rat central nervous system (Perry and Snyder, 1984;Kariya et al., 1985). Bradykinin B2 receptors have beenobserved in homogenates from the pons, medulla oblon-gata (Fujiwara et al., 1989), cerebral cortex, hippocampus,and spinal cord of the guinea pig brain (Fujiwara et al.,

Abbreviations

3 oculomotor nucleus6 abducens nucleus10 dorsal motor nucleus of the vagus12 hypoglossal nucleusA5 A5 noradrenaline cellsAcb nucleus accumbensAP area postremaAPT anterior pretectal nucleusC6 cervical segment 6 of the spinal cordCA CAfield of Ammon’s horncc central canalCd caudate nucleusChP choroid plexusCu cuneate nucleusDC dorsal cochlear nucleusdh dorsal hornDk nucleus DarkschewitschDLG dorsal lateral geniculate nucleusdlt dorsolateral tractDR dorsal rapheDRG, drg dorsal root ganglionDTg dorsal tegmentalECu external cuneate nucleusEP1 external plexiform layer of the olfactory bulbfr fasciculus retrofluxGe5 gelatinous layer of the caudal spinal trigeminal nucleusGi gigantocellular reticular nucleusG1 glomerular layer of the olfactory bulbGP globus pallidusGr gracile nucleusGrCb granular layer of the cerebellumGrDG granular layer of the dentate gyrusGrO granular layer of the olfactory bulbic internal capsuleIC inferior colliculusIMLF interstitial nucleus of the medial longitudinal fasciculusInG intermediate gray layer of superior colliculusInWh intermediate white layer of the superior colliculusIO inferior oliveIRt intermediate reticular nucleusL1 lumbar segment 1 of the spinal cordLa lateral amygdaloid nucleusLG lateral geniculateLM lateral mammillary nucleusLP lateral posterior thalamic nucleusLRt lateral reticular nucleusLSO lateral superior oliveLVe lateral vestibular nucleusMA3 medial accessory oculomotor nucleusMB mammillary bodyMGD medial geniculate nucleus, dorsal

MGV medial geniculate nucleus, ventralMo5 motor trigeminal nucleusMolCb molecular layer of the cerebellumMVe medial vestibular nucleusOp optic nerve layer of the superior colliculusopt optic tractOT nucleus of the optic tractPa paraventricular hypothalamic nucleusPAG periaqueductal grayPCRt parvocellular reticular nucleusPi pineal glandPir piriform cortexPLGL pleoglial periaqueductal grayPn pontine nucleusPnO pontine nucleus, oral partPo posterior thalamic nucleusPoDG polymorph layer of the dentate gyrusPPTg pedunculopontine tegmental nucleusPr5DM principle sensory trigeminal nucleus, dorsolateral partPr5VL principle sensory trigeminal nucleus, ventrolateral partPSol parasolitary nucleusPu putamenR red nucleusRt reticular thalamic nucleusRVL rostroventrolateral reticular nucleusS subiculumS2 sacral segment 2 of the spinal cordSCO subcommissural organSFO subfornical organSNC substantia nigra, caudalSNL substantia nigra, lateralSNR substantia nigra, reticular partSol nucleus of the solitary tractSolG gelatinous nucleus of the solitary tractSp5C caudal nucleus of the spinal trigeminalSp5O spinal trigeminal nucleus, oral partSPO superior paraolivary nucleusSpVe spinal vestibular nucleusSTh subthalamic nucleusSuG superficial gray layer of the superior colliculusSuVe superior vestibular nucleusT2 thoracic segment 2 of the spinal cordvh ventral hornVLG ventrolateral geniculate nucleusVLL ventral nucleus of the lateral lemniscusVPL ventroposterolateral thalamic nucleusVPM ventroposteromedial thalamic nucleusVTg ventral tegmental nucleusZI zona incertaZo zonal layer of the superior colliculus

204 C. MURONE ET AL.

1989; Sharif and Whiting, 1991). More specifically, brady-kinin B2 receptors have been localized to the nucleus of thesolitary tract, dorsal motor nucleus of the vagus, areapostrema, and spinal trigeminal nucleus of the guinea pigmedulla oblongata (Privitera et al., 1991); to the substan-tia gelatinosa of the dorsal horn in guinea pig and ratspinal cord (Lopes et al., 1993a, 1995); and to the dorsalroot ganglia of the guinea pig spinal cord (Steranka et al.,1988). Only these latter studies have localized bradykininB2 receptors to specific nuclei in the central nervoussystem. Taken together, these findings suggest the exis-tence of a functional endogenous kallikrein-kinin systemin the central nervous system. This study uses in vitroautoradiography with a radiolabelled bradykinin B2 recep-tor antagonist analogue, [125I]HPP-HOE140, (3-4-hydroxy-phenyl-propionyl-D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]bradyki-nin), which has been shown previously to have a highaffinity and specificity for the bradykinin B2 receptor (AbdAlla et al., 1993; Murone et al., 1996), to systemically mapthe distribution and characterize the bradykinin B2 recep-tor subtype in sheep brain and spinal cord and greatlyextends the known distribution of this receptor.

MATERIALS AND METHODS

Radioligand

The radioligand used to label bradykinin B2 receptorswas 3-4-hydroxyphenyl-propionyl-D-Arg0-[Hyp3, Thi5,D-Tic7,Oic8]bradykinin (HPP-HOE 140), a specific bradyki-nin B2 receptor antagonist analogue (Abd Alla et al., 1993;Murone et al., 1996). This compound was radioiodinatedby using the chloramine Tmethod (Greenwood et al., 1963)and was purified on a Sep-pak C18 cartridge by elutionwith a gradient of methanol (20–80%) in 0.1% trifluoroace-tic acid, giving an approximate specific activity of 820µCi/µg.

In vitro autoradiography

Brain tissues. Sheep brains (n 5 3) obtained fromfreshly killed animals at an abattoir were excised from theskull and transported on ice to the laboratory, where theywere blocked, and tissues were frozen in isopentane cooledon dry ice.Spinal cord tissues. Alaminectomywas performed on

sheep (n 5 2) killed with a lethal dose of sodium pentobar-bitone (100 mg/kg). The cord was removed within 60minutes and was kept on ice, during which time segmentsand their associated dorsal root ganglia were blocked andfrozen together in isopentane cooled on dry ice. Definitionof the segments was determined by the exit of the dorsalrootlets. The experiments were approved by the AnimalExperiment Ethics Committee of the Howard Florey Insti-tute of Experimental Physiology and Medicine, whichadheres to theAustralian Code of Practices.Brain and spinal cord tissues were stored at280°C until

sectioning, when coronal sections (10 µm and 20 µm) werecut in a cryostat maintained at 220°C. The sections werethaw mounted onto chrome alum-gelatine-coated slidesand dried over silica gel under vacuum at 4°C overnightprior to storage at 220°C. Alternate sections were used forautoradiography and staining with thionin for histologicalidentification of brain nuclei.Prior to incubation, the sections were equilibrated to

room temperature and then preincubated for 15 minutes

at 22°C in 170 mM Tris-HCl buffer, pH 7.4, containing0.2% bovine serum albumin (buffer A). The sections werethen transferred into a fresh volume of buffer containing,0.2 nM of [125I]HPP-HOE140 for 24 hours at 4°C. Nonspe-cific binding was determined in parallel incubations con-taining 1 µM HOE 140. At the completion of the incuba-tion, slides were transferred through four successive2-minute washes in a 170 mM Tris-HCl buffer, pH 7.4,without bovine serum albumin at 0°C. The slides werethen dried under a stream of cool air, loaded into x-raycassettes, and exposed to Kodak EB-1 x-ray film. [125I]radioactivity standards were prepared by applying knownamounts of [125I] radioactivity to 10- or 20-µm-thick,5-mm-diameter discs of brain sections mounted onto gela-tine-coated slides. These were exposed along with theincubated slides to allow for the quantitation of receptordensity using an MCID image analysis system (ImagingResearch Inc., Ontario, Canada). After 6 days of exposure,the films were developed in a Kodak RPX-Omat automaticprocessor.

Ligand specificity

[125I]HPP-HOE140 binding specificity to sheep spinalcord sections was determined by competition studies withbradykinin, the specific bradykinin B2 receptor antago-nists HOE140 and D-Arg[Hyp3,D-Phe 7,Leu8]bradykinin,and the B1 receptor antagonist, des-Arg9[Leu8]bradykinin.Serial sections of sheep spinal cord cervical segment 6 (C6)were incubated with at least 20 different concentrations(two sections per concentration) of each unlabelled brady-kinin analogue, ranging from 1025 M to 10214 M, in bufferA, containing 1 µM concentrations of the inhibitors leupep-tin, lisinopril, phosphoramidon, Plummer’s inhibitor (2-mercaptomethyl-3-guanidoethyl thiopropanoic acid), be-statin, and 1 mM benzamidine, and 2.5 mM 1,10phenanthroline to ensure that competing compounds re-main undegraded.

RESULTS

Ligand specificity

Competition of [125I]HPP-HOE140 binding to laminae IIand X of cervical segment 6 by HOE140, D-Arg[Hyp3,D-Phe7,Leu8]bradykinin and bradykinin are shown in Figure1a,b. For both areas, the rank order of affinities for thecompeting compounds were bradykinin . HOE140 :D-Arg[Hyp3,D-Phe7,Leu8]bradykinin. These compoundscompete for radioligand binding with apparent Ki values of0.32, 1.37, and 156 nM, respectively, at the lamina II siteand 0.13, 0.85, and 269, respectively, at the lamina X site. Thebradykinin B1 receptor antagonist des-Arg9[Leu8]bradykininfailed to compete for radioligand binding even at concentra-tions up to 10 µM.

Autoradiographic localization

The addition of 1 µMHOE140 to the incubation mediumcompletely blocked the binding of [125I]HPP-HOE140 in allareas except the amygdalohippocampal area and the cere-bral cortices, where 50–60% of the binding was nonspe-cific. In other regions, nonspecific binding was undetect-able.When determining the relative densities from selectedbrain regions (Table 1), the nonspecific binding was sub-tracted from the total binding, and only the specificdensities are shown. A detailed display of bradykinin B2

LOCALIZATION OF BRADYKININ B2 RECEPTORS 205

receptors is presented in Figures 2–8 along with adjacentthionin-stained sections. A similar pattern of distributionand density of receptors was detected in all of the nucleiand regions of the three sheep brains and two spinal cordsstudied. All neuroanatomical abbreviations in Figures 2–7are in accordance with the atlases of Paxinos and Watson(1986) and Paxinos and Huang (1995). As a guide, a highdensity of receptors is defined by a quantitative valuegreater than 190 dpm/mm2, whereas values between 95and 190 dpm/mm2 are moderate levels of bradykinin B2receptors, and levels below 95 dpm/mm2 are deemed lowlevels of binding.Olfactory bulb. A low density of bradykinin B2 recep-

tors are present in the glomerular layer of the olfactory

bulb, whereas no [125I]HPP-HOE140 binding is evident inthe external plexiform layer (Fig. 2B).Circumventricular organs. Interestingly, bradykinin

B2 receptors are detected in all of the circumventricularorgans. The most striking levels of receptors are in thesubfornical organ (Figs. 2E, 3D), the subcommissuralorgan (Fig. 4F,H), and area postrema (Fig. 7B), whereasmoderate levels are detected in the choroid plexus (Fig.3F), the pineal gland (Figs. 2H, 4F), the vascular organ ofthe lamina terminalis, and the median eminence (notshown).Basal ganglia. A low density of specific bradykinin B2

receptors are localized to the caudate nucleus (Figs. 2E,3B,D). A slightly lower density of binding sites with apatchy distribution are detected in the globus pallidus(Fig. 3D), the putamen (Figs. 2E, 3B), and the striatalbridges, which traverse the internal capsule linking thecaudate nucleus to the putamen; however, the internalcapsule itself is devoid of binding (Fig. 2E). A moderatedensity of bradykinin B2 binding sites is also observed inthe subthalamic nucleus (Fig. 4H) and the reticular part(Fig. 5B) of the substantia nigra, with a slightly lowerdensity of receptors visible over the compact (Fig. 5D) andlateral (Figs. 3H, 5B) components of the substantia nigra.Hypothalamus. A moderate density of binding is de-

tected in the lateral mammillary nucleus (Fig. 4H); how-

Fig. 1. Competition of [125I]3-4-hydroxyphenyl-propionyl-D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (HPP-HOE140) binding to laminaII (a) and lamina X (b) of sheep spinal cord cervical segment 6 (C6) bybradykinin and its analogues: circles, bradykinin; solid squares,HOE140; triangles, D-Arg[Hyp3,D-Phe7,Leu8 ]bradykinin; opensquares, des-Arg9[Leu8]bradykinin. At least 20 different concentra-tions of each unlabelled bradykinin analogue and bradykinin itselfwere assessed in duplicate. % B/BO, counts per minute of bound ligandover counts per minute of total ligand expressed as a percentage; [M],molar concentration.

TABLE 1. Relative Densities of [125I]HPP-HOE140 Binding to DifferentRegions of the Sheep Brain1

Region dpm/mm2

Circumventricular organsArea postrema 198 6 3Choroid plexus 115 6 13Pineal gland 181 6 3Subcommissural organ 190 6 9Subfornical organ 223 6 44

Basal gangliaCaudate nucleus 39 6 5Putamen 30 6 6Globus pallidus 19 6 5Substantia nigra, reticular part 119 6 13Subthalamic nucleus 142 6 8

HippocampusPolymorph layer of the dentate gyrus 43 6 16Granular layer of the dentate gyrus 25 6 5CA field of Ammon’s horn 39 6 10

HypothalamusParaventricular nucleus 26 6 8

ThalamusLateral posterior thalamic nucleus 87 6 3Ventroposterolateral thalamic nucleus 103 6 0.6Medial habenula nucleus 52 6 6Zona incerta 120 6 19

MidbrainPeriaqueductal gray 87 6 3Superior colliculus 131 6 14Inferior colliculus 94 6 52Oculomotor nucleus 297 6 60Trochlear nucleus 289 6 9Pleoglial periaqueductal gray 289 6 25Amygdala 71 6 2

HindbrainDorsal tegmental nucleus 120 6 4Pontine nucleus 95 6 8Pontine nucleus, oral part 86 6 3Parasolitary nucleus 108 6 11Nucleus of the solitary tract 95 6 0.7Inferior olive 105 6 4Spinal trigeminal nucleus 139 6 13Parvocellular reticular nucleus 75 6 8Intermediate reticular nucleus 32 6 4

CerebellumMolecular layer 159 6 22

CortexPiriform 23 6 3

1Mean 6 S.D. of values derived from at least three sections. Only specific densities areshown.


Fig. 2. A–L: Distribution of bradykinin B2 binding sites in thesheep central nervous system demonstrated by in vitro autoradiogra-phy employing [125I]HPP-HOE140 as the radioligand. The photomicro-graphs on the left show sections stained with thionin. The autoradio-graphs in themiddle show adjacent sections depicting total bradykininB2 receptor binding. The autoradiographs on the right show bradyki-

nin B2 receptor binding in the presence of 1 µMHOE140 and representnonspecific binding. The white areas in the autoradiographs representareas of radioligand binding. The figures are representative sectionsthrough the central nervous system and proceed from rostral tocaudal. For abbreviations, see list. Scale bar inAalso applies to B, C; Dalso applies to E–I; J also applies to K,L.


Fig. 3. A–H: Distribution of bradykinin B2 binding sites in thesheep forebrain demonstrated by in vitro autoradiography employing[125I]HPP-HOE140 as the radioligand. The photomicrographs on theleft show sections stained with thionin. The autoradiographs on theright show adjacent sections depicting total bradykinin B2 receptor

binding. The white areas in the autoradiographs represent areas ofradioligand binding. Nonspecific binding, determined in the presenceof 1 µM HOE140, produced no detectable image; therefore, the imagesin B, D, F, and H represent specific binding. The figures proceed fromrostral to caudal. For abbreviations, see list.

Fig. 4. A–H: Distribution of bradykinin B2 binding sites in thesheep forebrain and midbrain demonstrated by in vitro autoradiogra-phy employing [125I]HPP-HOE140 as the radioligand. The photomicro-graphs on the left show sections stained with thionin. The autoradio-graphs on the right show adjacent sections depicting total bradykininB2 receptor binding. The white areas in the autoradiographic panels

represent areas of radioligand binding. Nonspecific binding, deter-mined in the presence of 1 µMHOE140, produced no detectable image;therefore, the images in B, D, F, and H represent specific binding. Thefigures proceed from rostral to caudal. For abbreviations, see list.Scale bar inA also applies to B–D, E also applies to F–H.

Fig. 5. A–H: Distribution of bradykinin B2 binding sites in thesheep midbrain demonstrated by in vitro autoradiography employing[125 I]HPP-HOE140 as the radioligand. The photomicrographs on theleft show sections stained with thionin. The autoradiographs on theright show adjacent sections depicting total bradykinin B2 receptor



Fig. 6. A–H: Distribution of bradykinin B2 binding sites in thesheep midbrain and hindbrain demonstrated by in vitro autoradiogra-phy employing [125I]HPP-HOE140 as the radioligand. The photomicro-graphs on the left show sections stained with thionin. The autoradio-graphs on the right show adjacent sections depicting total bradykinin

B2 receptor binding. The white areas in the autoradiographs representareas of radioligand binding. Nonspecific binding, determined in thepresence of 1 µM HOE140, produced no detectable image; therefore,the images in B, D, F, and H represent specific binding. The figuresproceed from rostral to caudal. For abbreviations, see list.


Fig. 7. A–H: Distribution of bradykinin B2 binding sites in thesheep hindbrain demonstrated by in vitro autoradiography employing[125I]HPP-HOE140 as the radioligand. The photomicrographs on theleft show sections stained with thionin. The autoradiographs on theright show adjacent sections depicting total bradykinin B2 receptor



Fig. 8. A–H: Distribution of bradykinin B2 receptors in the differ-ent regions of the sheep spinal cord demonstrated by in vitro autoradi-ography employing [125I]HPP-HOE140 as the radioligand. The photo-micrographs on the left show thionin stained sections. Theautoradiographs on the right show adjacent sections, the white areas

depicting total bradykinin B2 receptor binding. Nonspecific binding,determined in the presence of 1 µM HOE140, produced no detectableimage; therefore, the images in B, D, F, and H represent specificbinding. For abbreviations, see list.


ever, the mammillothalamic tract is devoid of binding,whereas the paraventricular nucleus (Fig. 3D) displays alow level of bradykinin B2 receptors.Amygdala and hippocampal formation. The lateral

amygdala (Fig. 3F), amygdalohippocampal area, poly-morph and granular layers of the dentate gyrus, and theCA field of Ammon’s horn (Fig. 4D) all display a diffusepattern of low-density binding.Thalamus. Both the lateral (Fig. 3F) andmedial (Figs.

2H, 3F, 4F) portions of the ventroposterior thalamic nucleiexhibit a diffuse and moderate density of bradykinin B2receptors, as does the zona incerta (Figs. 3F,H, 4F,H). Thislevel of binding is also visible over the reticular thalamicnucleus (Fig. 2H); however, in this nucleus, the binding ispunctate. The medial and lateral habenular nuclei (notshown) and the lateral (Figs. 2H, 3H, 4F) and posterior(Fig. 4F) portions of the posterior thalamic nuclei alldisplay a low level of bradykinin B2 receptors.Midbrain. The oculomotor (Fig. 5D,F), trochlear, and

red nuclei (Fig. 5F) show strikingly high levels of bradyki-nin B2 receptors in the midbrain, as does the pleoglialperiaqueductal gray (Fig. 5B), whereas moderate levelsare detected in the magnocellular portion of the rednucleus and the pedunculopontine tegmental nucleus (Fig.5H). Low levels of binding are detected over the periaque-ductal gray (Fig. 5F), the dorsal (Fig. 5H) and medianraphe nuclei, and the parabigeminal nucleus (not shown).Visual system. A moderate density of binding is ob-

served over the dorsal lateral (Figs. 2H, 4F) and ventrallateral (Fig. 4F) geniculate nuclei. Both the olivary pretec-tal and the anterior pretectal nuclei (Figs. 4F,H, 5B) showa moderate density of binding, as does the Darkschewitschnucleus (Figs. 4H, 5B). Interestingly, a low level of bindingis detected over the rostral interstitial nucleus of themedial longitudinal fasciculus; however, the caudal inter-stitial nucleus of the medial longitudinal fasciculus (Fig.5D) is devoid of any binding. The zonal and superficial graylayers of the superior colliculus (Fig. 5D) display moderatelevels of bradykinin B2 receptors, whereas the intermedi-ate gray and white layers have a low density of receptors.The dorsomedial optic nerve layer (Fig. 5B,D) is conspicu-ous by its lack of any receptor binding. Interestingly,binding becomes visible and more intense along the opticnerve layer and the intermediate white layer as the layersmove ventrolaterally. However, it is difficult to attributethis binding to these layers; more likely, this binding is tothe nucleus of the optic tract (Fig. 4F, 5B) and the posteriorpretectal nucleus, respectively.Auditory system. A moderate density of binding is

localized to the ventral medial geniculate nucleus (Figs.4H, 5B) and the caudal nucleus of the inferior colliculus.The dorsal (Fig. 5B) and medial portions of the medialgeniculate and the external and dorsal cortices of theinferior colliculus all display low levels of bradykinin B2receptors.Hindbrain. Moderate densities of bradykinin B2 recep-

tors are detected in the dorsal and ventral tegmentalnuclei (Fig. 6B) and also in the pontine nucleus (Fig. 6B,D);however, the oral part (Fig. 5B) of the pontine nucleusdisplays a very diffuse, patchy, and low density of recep-tors. In areas occurring more caudally, moderate to highdensities of receptors are detected in the dorsal cochlearnucleus, the abducens nucleus, the motor trigeminalnucleus, the ventral and dorsal principle sensory trigemi-nal nuclei, the superior paraolivary nucleus, and the

lateral superior olive (Fig. 6D,F). The superior (Fig. 6D),medial, lateral, and spinal vestibular nuclei (Fig. 6F) alldisplay moderate levels of bradykinin B2 receptors. Thereis also a moderate amount of binding over the regionencompassing the A5 noradrenaline cells; however, somehigh-power emulsion autoradiography needs to be under-taken to determine whether this binding is directly onthese cells.The nucleus of the solitary tract (Fig. 6H) also displays

moderate levels of bradykinin B2 receptors, as do theparasolitary nucleus (Fig. 6H) and the gelatinous nucleusof the solitary tract (Figs. 6H, 7B). Interestingly, thebinding density increases dramatically in the gelatinousnucleus of the solitary tract as the nucleus extends ros-trally. Similarly, the gelatinous layer of the caudal spinaltrigeminal nucleus (Fig. 7B,D,F) displays intense binding,whereas the spinal trigeminal nucleus, in its caudalportion and oral part (Fig. 6F), only displays a moderatedensity of binding. Of further interest, the distribution ofthis receptor on the spinal trigeminal nucleus is quitepatchy. Areas that also display a moderate density ofbinding in the hindbrain are the inferior olive (Figs. 2K,7D) and the cuneate (Fig. 7B,D) and external cuneate(Figs. 2K, 6H) nuclei, whereas low levels are detected inthe hypoglossal (Fig. 7B) and gracile nuclei (Fig. 7B). Nobradykinin B2 receptors are detected over the dorsal motornucleus of the vagus (Fig. 6H, 7B) or the nucleus am-biguus. The pyramidal tract and the spinal trigeminaltract are also devoid of binding.Reticular formation. Alow level of binding is detected

in the intermediate, parvocellular, gigantocellular (Fig.2K), and rostral ventrolateral (Fig. 6H) reticular nuclei.Only the lateral reticular nucleus (Fig. 7D) displays aslightly higher density of receptors.Cerebellar and cerebral cortices. In the cerebellum,

a diffuse, moderate density of binding is detected through-out the molecular layer (Fig. 7H). The cerebral corticiesdisplay low levels of binding, which are spread diffuselythroughout its layers.Spinal cord. A similar distribution of bradykinin B2

receptors is detected across all of the cervical (C), thoracic(T), lumbar (L), and sacral (S) segments of the spinal cordstudied (Fig. 8). The most intense binding occurs in laminaX, with only a slight decrease in intensity in laminae I andII of the dorsal horn; however, the intensity of the bindingin lamina II is higher in the sacral segment compared withthat in the lumbar segment, whereas the intensity ofbinding in lamina X is lower in the sacral and thoracicsegments compared with that in the cervical segment(Table 2). The dorsal root ganglia associated to each of thesegments studied display an equally high density of bind-ing in the cervical and thoracic segments but lower thanthat in the lumbar segment, and this binding is quitepatchy. The dorsal root ganglion associated with the sacralsegment was not available for autoradiographic quantita-tion. Binding is also detected in the dorsolateral tract at amoderate level. Low levels of binding are detected through-out the remaining gray matter, whereas no binding isdetected throughout the whitematter nor the intermedola-teral column of the thoracic segments.

DISCUSSION

The development of a second generation of more potentand specific antagonists of the bradykinin receptors, such


as HOE 140, which contains several synthetic amino acidsubstitutions (Hock et al., 1991; Rhaleb et al., 1992) and isa selective and potent bradykinin B2 receptor antagonist(Bao et al., 1991; Hock et al., 1991; Wirth et al., 1991;Rhaleb et al., 1992), has enabled the delineation andcharacterization of the bradykinin B2 receptor protein byaffinity cross-linking studies (Abd Alla et al., 1993). HOE140 displays higher affinity and specificity than previousbradykinin B2 receptor antagonists (Togo et al., 1989;Regoli et al., 1990; Rhaleb et al., 1991), which are limitedby their partial agonist activity and are subject to degrada-tion by proteolytic enzymes (Regoli et al., 1990; Dray andPerkins, 1993).In this study, we have mapped the distribution of the

bradykinin B2 receptor by using [125I]HPP-HOE140, ananalogue of the specific bradykinin B2 receptor antagonist.We have previously shown that this radioligand is specificand has a high affinity for the bradykinin B2 receptor(Murone et al., 1996). Using sheep spinal cord sections, therank order of affinity of the bradykinin analogues for thebradykinin B2 receptor is bradykinin . HOE140 :D-Arg[Hyp3,D-Phe7,Leu8]bradykinin. The bradykinin B1receptor antagonist des-Arg9[Leu8]bradykinin does notcompete for [125I]HPP-HOE140 binding at concentrationsup to 10 µM, adding further support to the finding that thenature of this receptor is of the B2 subtype. A previousstudy that used [125I][Tyr8]bradykinin to localize receptorsin the guinea pig hindbrain reported a Ki of ,0.1 nM incompetition experiments using bradykinin to competewith the radioligand (Privitera et al., 1991). Similarly, inthe guinea pig lumbar spinal cord (Lopes et al., 1993a) andin the rat lumbar spinal cord (Lopes et al., 1995), Ki valuesranging from 1 nM to 40 nM and from 0.01 nM to 0.1 nM,respectively, were determined for a range of bradykinin B2receptor antagonists used to compete for binding with[125I][Tyr8]bradykinin in the lamina II region. These affini-ties are comparable to our findings in which bradykininand HOE140 compete for [125I]HPP-HOE140 binding withKi values of ,0.3 nM and 1.4 nM, respectively.Previously, the bradykinin B2 receptor has been local-

ized to particular nuclei only in the rat and guinea pigspinal cord (Steranka et al., 1988; Lopes et al., 1993a,1995) and in the guinea pig hindbrain (Privitera et al.,1991). Here, we present for the first time a detailed map ofthe occurrence of the bradykinin B2 receptor in the sheepbrain and spinal cord. Our data indicate that this receptoris extensively distributed throughout motor and sensoryareas of the sheep central nervous system and is present inmidbrain and forebrain structures that have not beenpreviously reported in any species.The overall pattern of binding in the sheep spinal cord

and dorsal root ganglia resembles that of the guinea pigspinal cord (Lopes et al., 1993a) and dorsal root ganglia(Steranka et al., 1988), where bradykinin B2 receptors are

detected over laminae I and II of the dorsal horn and thedorsal root ganglia. The interesting difference we find isthe striking binding in the laminae X of the spinal cord, anarea that is also involved in the relay of sensory informa-tion (Martin and Jessell, 1991); however, a function forbradykinin in this area remains to be determined. Nocicep-tive fibres are known to project to laminae I and II of thedorsal horn (Jessell and Kelley, 1991). The severing ofthese fibres by dorsal rhizotomy results in a significantdecrease of bradykinin B2 receptors in the ipsilateraldorsal horn, particularly in lamina II, suggesting thatthese receptors are present presynaptically (Lopes et al.,1993a, 1995; Murone et al., unpublished observation). Ourfindings support earlier functional studies in which admin-istration of bradykinin intrathecally increased reactiontime to noxious stimuli (Laneuville and Couture, 1987)and also increased arterial pressure and decreased heartrate in rats (Lopes et al., 1993b). Interestingly, intraarte-rial injections of bradykinin into rats induced pain thatwas blocked by coadministration with a bradykinin B2receptor antagonist (Steranka et al., 1988), whereas intra-cerebroventricular administration of bradykinin causedan anxiogenic effect in rats (Bhattacharya et al., 1995) andan antinociceptive effect in rabbits (Ribeiro and Rocha eSilva, 1973). These observations and the presence ofbradykinin B2 receptors in laminae I, II, and X of thespinal cord and the dorsal root ganglia may support a rolefor bradykinin as a modulator in the sensory pathway.Furthermore, microinjection of bradykinin into the dorsalperiaqueductal gray induced hyperalgesia, which is antago-nized by naloxone, suggesting that an opioid mediates thiscentral effect of bradykinin (Burdin et al., 1992). In thecurrent study, a low but significant binding is detected inthe periaqueductal gray, which may suggest that bradyki-nin via these receptors mediates the local release of opioidsto induce hyperalgesia. We also detect a moderate densityof binding in the ventroposterior thalamic nuclei and thecaudal nucleus of the spinal trigeminal. These are nucleithat belong to a pathway involved in the relay of tactileinformation from the facial nerves (Dodd and Kelley,1991). This thalamic localization is similar to our earlierfindings in the guinea pig (Murone et al., 1996).Along with the well-known actions of bradykinin in the

sensory pathway are its actions in cardiovascular homeo-stasis. A role for bradykinin in cardiovascular control issupported by the actions observed when the peptide isadministered centrally. Injection of bradykinin directlyinto the nucleus of the solitary tract of awake rats resultedin a pressor response (Fior et al., 1993). More recently,microinjection of bradykinin into the nucleus of the soli-tary tract of anaesthetized rats produced hypotension andbradycardia, and these effects were attenuated by adminis-tration of HOE140 or des-Arg9[Leu8]bradykinin, suggest-ing that both bradykinin B2 and B1 receptorsmay facilitatethese cardiovascular effects (Caligiorne et al., 1996). Thisgroup goes on to suggest that the surgical procedures usedto perform the microinjection could have induced thebradykinin B2 receptor subtype or, alternatively, the anaes-thetic may have interfered with the response, because ithas been suggested that the effects of intracerebroventricu-larly administered kinins may differ depending on thepresence of anaesthesia (Diz, 1985). Ultimately, thesestudies describe the modulation of the cardiovascularsystem by bradykinin injected into the nucleus of thesolitary tract and, taken together with the localization of

TABLE 2. Relative Densities of [125I]HPP-HOE140 Binding in DifferentRegions and Segments of Spinal Cord1

Region C6 T2 L3 S2

LI/II 254 6 27 216 6 43 202 6 32 279 6 30LX 305 6 7 240 6 23 245 6 35 236 6 16DRG 245 6 20 223 6 14 178 6 21 ND

1Mean 6 S.D. of values (dpm/mm2) taken from four different sections. ND, notdetermined; C, cervical; L, lumbar; S, sacral; T, thoracic; LI/LII, laminae I–II of thedorsal horn of the spinal cord; LX, lamina X of the spinal cord; DRG, dorsal rootganglion.


bradykinin B2 receptors in this region reported in thisstudy and others (Privitera et al., 1991; Murone et al.,1996), suggest that these receptors may mediate thecardiovascular effects. Interestingly, in this study, we alsodetect bradykinin B2 receptors in the gelatinous nucleus ofthe solitary tract, which is part of the gustatory system(Norgren, 1995; Saper, 1995); however, the exact functionof bradykinin in this subnucleus remains to be deter-mined.Our results also show that many of the circumventricu-

lar organs display high densities of bradykinin B2 recep-tors. One of the distinguishing characteristics of thecircumventricular organs is that all but the subcommis-sural organ lack a blood-brain barrier, allowing access ofcirculating hormones where they may then exert theireffects on the central nervous system via specific receptorsin the circumventricular organs. (McKinley et al., 1990).Early studies have reported that intracarotid infusion ofbradykinin produced hypotension in anaesthetized cats(Rocha e Silva et al., 1960). Subsequent studies reportedthat bradykinin infusion via the same route produced aninitial decrease in blood pressure followed by an hyperten-sive response (Lang and Pearson, 1968; Pearson et al.,1969); conversely, a pressor response was reported inanaesthetized dogs (Wilkinson and Scroop, 1985). Further-more, the presence of both kininogenase and glandularkallikrein-like immunoreactivity in human cerebrospinalfluid (Scicli et al., 1984) suggests that the pressor responsemay be regulated by locally generated kinins acting oncircumventricular organs. Intracerebroventricularly ad-ministered kinins elicit a wide variety of effects includinghypertension and bradycardia (Diz, 1985; Lindsey et al.,1989; Privitera, 1992; Madeddu et al., 1994), antinocicep-tion (Ribeiro et al., 1971; Laneuville et al., 1989), andantidiuresis (Hoffman and Schmid, 1978). The antidiureticeffect elicited by bradykinin infusion is blocked by lesionsof the median eminence, suggesting that release of antidi-uretic hormone is the mediator of this effect (Hoffman andSchmid, 1978). We have shown that the median eminencedisplays a moderate density of bradykinin B2 receptorsthat maymediate the release of antidiuretic hormone.Alsoof interest is the intense binding we detect in the areapostrema, because intracarotid infusion of bradykininincreased blood pressure and heart rate, and this effectwas abolished by ablation of the area postrema (Wilkinsonand Scroop, 1985). A similar effect was described whenbradykinin was administered into the fourth ventricle(Lindsey et al., 1988), and this group suggests that theaction may be mediated by the area postrema.Precise sites in the hypothalamus that are responsive to

bradykinin have been identified and reported to producedifferential effects (Diz and Jacobowitz, 1984a,b). In therostral area of the hypothalamus, bradykinin microinjec-tion elicits an increase in blood pressure and heart rate,whereas, in the preoptic suprachiasmatic nucleus, heartrate is increased with no change in blood pressure. In theparaventricular nucleus, an area in which we have de-tected a low density of bradykinin B2 receptors, bradykininproduced a decrease in heart rate but did not alter bloodpressure (Diz and Jacobowitz, 1984b). Furthermore, brady-kinin-like immunoreactivity has been detected in thehypothalamus (Correa et al., 1979; Perry and Snyder,1984). These findings suggest that centrally producedbradykininmay act directly on the paraventricular nucleusto elicit these responses. No effect was detected when

bradykinin was microinjected in the caudal part of thehypothalamic nucleus (Diz and Jacobowitz, 1984a,b).An earlier study detected bradykinin B2 receptors in the

nucleus of the solitary tract, area postrema, spinal trigemi-nal nucleus, and dorsal motor nucleus of the vagus in theguinea pig hindbrain (Privitera et al., 1991). In our study,we extend this distribution of the bradykinin B2 receptorin the hindbrain, although we do not detect any binding inthe dorsal motor nucleus of the vagus. In particular, wedetect bradykinin B2 receptors in the external cuneate, theparvocellular reticular nucleus, the rostral ventrolateralreticular nucleus, and the spinal trigeminal nucleus. Thebinding in these areas is curious, because microinjection ofbradykinin into these particular nuclei did not elicit anypressor responses (Fior et al., 1992, 1993). However, anincrease in mean arterial pressure when bradykinin wasinjected into the rostral ventrolateral reticular nucleushas been reported (Privitera et al., 1994). Of furtherinterest is the striking density of bradykinin B2 receptorswe detected in the pleoglial periaqueductal gray, demon-strating that these receptors are found not only in neuronsbut also in glia.Our results demonstrate a wide distribution of bradyki-

nin B2 receptors in the sheep brain. The localization ofsome of these receptors is consistent with a role forbradykinin in modulating sensory and cardiovascularfunction; however, we also demonstrate a number ofregions, for example, in the basal ganglia, the visualsystem, the auditory system, and the more rostral hind-brain regions in which the actions of bradykinin remainunknown.

ACKNOWLEDGMENTS

We thank Drs. G. Breipohl, J. Knolle, K. Wirth, and B.Scholkens (Hoechst AG, Frankfurt, Germany) for the kindgifts of HOE 140 and HPP-HOE140 and D. Casley foriodinating the ligand, HPP-HOE140.

LITERATURE CITED

Abd Alla, S., J. Buschko, U. Quitterer, A. Maidhof, M. Haasemann, G.Breipohl, J. Knolle, and W. Muller-Esterl (1993) Structural features ofthe human bradykinin B2 receptor probed by agonists, antagonists, andanti-idiotypic antibodies. J. Biol. Chem. 268:17277–17285.

Bao, G., F. Qadri, B. Stauss, H. Stauss, P. Gohlke, and T. Unger (1991)HOE140, a new highly potent and long-acting bradykinin antagonist inconscious rats. Eur. J. Pharmacol. 200:179–182.

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

Bhattacharya, S.K., P.J.R. Mohan Rao, and A.P. Sen (1995) Anxiogenicactivity of intraventricularly administered bradykinin in rats. J. Psycho-pharmacol. 9:348–354.

Bhoola, K.D., C.D. Figueroa, and K. Worthy (1992) Bioregulation of kinins:Kallikreins, kininogens and kininases. Pharmacol. Rev. 44:1–80.

Burdin, T.A., F.G. Graeff, and I.R. Pela (1992) Opioid mediation of theantiaversive and hyperalgesic actions of bradykinin injected into thedorsal periaqueductal gray of the rat. Physiol. Behav. 52:405–410.

Caligiorne, S.M., R.A.S. Santos, and M.J. Campagnole-Santos (1996)Cardiovascular effects produced by bradykinin microinjection into thenucleus tractus solitarii of anaesthetized rats. Brain Res. 720:183–190.

Chao, J., L. Chao, C.C. Swain, J. Tsai, and H.S. Margolius (1987) Tissuekallikrein in rat brain and pituitary: Regional distribution and estrogeninduction in the anterior pituitary. Endocrinology 120:475–482.

Correa, F.M.A., R.B. Innis, G.R. Uhl, and S.H. Snyder (1979) Bradykinin-like immunoreactive neuronal systems localized histochemically in ratbrain. Proc. Natl. Acad. Sci. USA 76:1489–1493.


Diz, D.I. (1985) Bradykinin and related peptides in central control of thecardiovascular system. Peptides 6:57–64.

Diz, D.I., and D.M. Jacobowitz (1984a) Cardiovascular actions of fourneuropeptides in the rat hypothalamus. Clin. Exp. Theory Pract.A6:2085–2090.

Diz, D.I., and D.M. Jacobowitz (1984b) Cardiovascular effects of discreteintrahypothalamic and preoptic injections of bradykinin. Brain Res.Bull. 12:409–417.

Doctrow, S.R., S.M. Abelleira, L.A. Curry, R. Heller-Harrison, J.W. Kozar-ich, B. Malfroy, L.A. McCarroll, K.G. Morgan, A.R. Morrow, G.F. Musso,J.L. Smart, J.A. Straub, B. Turnbull, and C.A. Gloff (1994) Thebradykinin analogue RMP-7 increases intracellular free calcium levelsin rat brain microvascular endothelial cells. J. Pharmacol Exp. Ther.271(1):229–237.

Dodd, J., and J.P. Kelley (1991) Trigeminal System. In E.R. Kandel, J.H.Schwartz, and T.M. Jessell (eds): Principles in Neural Science. NewYork: Elsevier Science Publishing, pp. 701–710.

Dray, A., and M. Perkins (1993) Bradykinin and inflammatory pain. TrendsNeurosci. 16:99–104.

Fior, D.R., D.T.O. Martins, and C.J. Lindsey (1992) Tissue kallikrein-kininsystem in the brain and the regulation of arterial pressure. AgentsActions 38(Suppl. III):31–38.

Fior, D.R., D.T.O. Martins, and C.J. Lindsey (1993) Localization of centralpressor action of bradykinin in medulla oblongata. Am. J. Physiol265:H1000–H1006.

Fujiwara, Y., C.R. Mantione, R.J. Vavrek, J.M. Stewart, and H.I. Yama-mura (1989) Characterization of [3H]bradykinin binding sites in guineapig central nervous system: Possible existence of B2 subtypes. Life Sci.44:1645–1653.

Greenwood, F.C., W.M. Hunter, and J.S. Glover (1963) The preparation of131I-labelled human growth hormone of high specific radioactivity.Biochem. J. 89:114–123.

Hock, F.J., K. Wirth, U. Albus, W. Linz, H.J. Gerhards, G. Wiemer, S.Henke, G. Briepohl, W. Konig, J. Knolle, and B.A. Scholkens (1991)HOE140, a new potent and long-acting bradykinin antagonist: in vitrostudies. Br. J. Pharmacol. 102:769–773.

Hoffman, W.E., and P.G. Schmid (1978) Separation of pressor and antidi-uretic effects of intraventricular bradykinin. Neuropharmacology 17:999–1002.

Jessel, T.M., and D.D. Kelley (1991) Pain and analgesia. In E.R. Kandel,J.H. Schwartz, and T.M. Jessell (eds): Principles of Neural Science. NewYork: Elsevier Science Publishing, pp. 385–399.

Kariya, K., and A. Yamauchi (1981) Effects of intraventricular injection ofbradykinin on the EEG and the blood pressure in conscious rats.Neuropharmacology 20:1221–1224.

Kariya, K., A. Yamauchi, and T. Sasaki (1985) Regional distribution andcharacterization of kinin in the CNS of the rat. J. Neurochem. 44:1892–1897.

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

Laneuville, O., T.A. Reader, and R. Couture (1989) Intrathecal bradykininacts presynaptically in spinal noradrenergic terminals to produceantinociception in the rat. Eur. J. Pharmacol. 159:273–283.

Lang, W.J., and L. Pearson (1968) Studies on the pressor responsesproduced by bradykinin and kallidin. Br. J. Pharmacol. Chemother.32:330–338.

Lindsey, C.J., K. Fujita, and T.O. Martins (1988) The central pressor effectof bradykinin in normotensive and hypertensive rats. Hypertension11(Suppl. I):I126–I129.

Lindsey, C.J., C.R. Nakaie, and D.T.O. Martins (1989) Central nervoussystem kinin receptors and the hypertensive response mediated bybradykinin. Br. J. Pharmacol. 97:763–768.

Lopes, P., S. Kar, C. Tousignant, D. Regoli, R. Quirion, and R. Couture(1993a) Autoradiographic localization of [125I-Tyr8]bradykinin receptorbinding sites in the guinea pig spinal cord. Synapse 15:48–57.

Lopes, P., D. Regoli, and R. Couture (1993b) Cardiovascular effects ofintrathecally administered bradykinin in the rat: characterization ofreceptors with antagonists. Br. J. Pharmacol. 110:1369–1374.

Lopes, P., S. Kar, L. Chretien, D. Regoli, R. Quirion, and R. Couture (1995)Quantitative autoradiographic localization of [125I-Tyr8]bradykinin re-ceptor binding sites in the rat spinal cord: Effects of neonatal capsaicin,

noradrenergic deafferentation, dorsal rhizotomy and peripheral axotomy.Neuroscience 68:867–881.

Madeddu, P., N. Glorioso, A. Soro, G. Tonolo, P. Manuta, C. Troffa, M.P.Demontis, M.V. Varoni, and V. Anania (1990) Brain kinins are respon-sible for the pressor effect of intracerebroventricular captopril inspontaneously hypertensive rats. Hypertension 15:407–412.

Madeddu, P., N. Glorioso, M.V. Varoni, M.P. Demontis, M.C. Fattaccio, andV.Anania (1994) Cardiovascular effects of brain kinin receptor blockadein spontaneously hypertensive rats. Hypertension 23(Suppl. I):I189–I192.

Marceau, F. (1995) Kinin B1 receptors: A review. Immunopharmacology30:1–26.

Marceau, F., A. Lussier, D. Regoli, and J.P. Giroud (1983) Pharmacology ofkinins: Their relevance to tissue injury and inflammation. Gen. Pharma-col 14:209–229.

Martin, J.H., and T.M. Jessell (1991) Anatomy of the somatic sensorysystem. In E.R. Kandel, J.H. Schwartz, and T.M. Jessell (eds): Prin-ciples of Neural Science. New York: Elsevier Science Publishing, pp.353–366.

McKinley, M.J., R.M. McAllen, F.A.O. Mendelsohn, A.M. Allen, S.Y. Chai,and B.J. Oldfield (1990) Circumventricular organs: neuroendocrineinterfaces between the brain and the hemal milieu. Front. Neurodendo-crinol. 11:91–127.

Murone, C., R.B. Perich, I. Schlawe, S.Y. Chai, D. Casley, D.P. MacGregor,W. Muller-Esterl, and F.A.O. Mendelsohn (1996) Characterization andlocalization of bradykinin B2 receptors in the guinea pig using aradioiodinated HOE140 analogue. Eur. J. Pharmacol. 306:237–247.

Norgren, R. (1995) Gustatory system. In G. Paxinos (ed): The Rat NervousSystem. San Diego: Academic Press, Inc., pp. 751–772.

Okada, Y., Y. Tuchiya, M.Yagyu, S. Kozawa, and K. Kariya (1977) Synthesisof bradykinin fragments and their effect on pentobarbital sleeping timein mouse. Neuropharmacology 16:381–383.

Paxinos, G., and X.-F. Huang (1995) Atlas of the Human Brainstem. SanDiego: Academic Press, Inc.

Paxinos, G., and C. Watson (1986) The Rat Brain in Stereotaxic Coordi-nates. Sydney: Academic Press.

Pearson, L., G.A. Lambert, and W.J. Lang (1969) Centrally mediatedcardiovascular and EEG responses to bradykinin and eledoisin. Eur. J.Pharmacol. 8:153–158.

Perry, D.C., and S.H. Snyder (1984) Identification of bradykinin inmamma-lian brain. J. Neurochem. 43:1073–1080.

Privitera, P.J. (1992) Brain kallikrein-kinin system in arterial pressureregulation. AgentsActions 38(Suppl. III):39–46.

Privitera, P.J., P.R. Daum, D.R. Hill, and C.R. Hiley (1991) Autoradio-graphic visualization and characteristics of [125I]bradykinin bindingsites in guinea pig brain. Brain Res. 577:73–79.

Privitera, P.J., H. Thibodeaux, and P. Yates (1994) Rostal ventrolateralmedulla as a site for the central hypertensive action of kinins. Hyperten-sion 23:52–58.

Regoli, D., and J. Barabe (1980) Pharmacology of bradykinin and relatedkinins. Pharmacol. Rev. 32:1–46.

Regoli, D., N.-E. Rhaleb, S. Dion, and G. Drapeau (1990) New selectivebradykinin receptor antagonists with bradykinin B2 receptor character-ization. Trends Pharmacol. Sci. 11:156–161.

Rhaleb, N.-E., S. Telemaque, N. Rouissi, S. Dion, D. Jukic, G. Drapeau, andD. Regoli (1991) Structure-activity studies of bradykinin and relatedpeptides. Hypertension 17:107–115.

Rhaleb, N.-E., N. Rouissi, D. Jukic, D. Regoli, S. Henke, G. Breipohl, and J.Knolle (1992) Pharmacological characterization of a new highly potentB2 receptor antagonsit (HOE140: D-Arg-[Hyp3,Thi5,D-Tic7,Oic8] brady-kinin). Eur. J. Pharmacol. 210:115–120.

Ribeiro, S.A., and M. Rocha e Silva (1973) Antinociceptive action ofbradykinin and related kinins of larger molecular weights by theintraventricular route. Br. J. Pharmacol. 47:517–528.

Ribeiro, S.A., A.P. Corrado, and F.G. Graeff (1971) Antinociceptive action ofintraventricular bradykinin. Neuropharmacology 10:725–731.

Rocha e Silva, M., A.P. Corrado, and A.O. Ramos (1960) Potentiation ofduration of the vasodilator effect of bradykinin by sympatholytic drugs.J. Pharmacol. Exp. Ther. 128:217–226.

Saper, C.B. (1995) Central autononomic system. In G. Paxinos (ed): The RatNervous System. San Diego: Academic Press, Inc., pp. 107–136.


Scicli, A.G., G. Forbes, H.L. Nolly, M. Dujovny, and O.A. Carretero (1984)Kallikrein-kinins in the central nervous system. Clin. Exp. TheoryPract. A6:1731–1738.

Sharif, N.A., and R.L. Whiting (1991) Identification of B2 bradykininreceptors in guinea pig brain regions, spinal cord and peripheraltissues. Neurochem. Int. 18:89–96.

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

Togo, J., R.M. Burch, C.J. DeHaas, J.R. Connor, and L.R. Steranka (1989)

D-Phe7 substituted peptide bradykinin antagonists are not substratesfor kininase II. Peptides 19:109–112.

Wiemer, G., R. Popp, B.A. Scholkens, and H. Gogelein (1994) Enhancementof cytosolic calcium, prostacyclin and nitic oxide by bradykinin and theACE inhibitor ramiprilat in porcine brain capillary endothelial cells.Brain Res. 638(1-2):261–266.

Wilkinson, D.L., and G.C. Scroop (1985) Role of prostaglandins and theareas postrema in the central pressor action of bradykinin. Eur. J.Pharmacol. 113:287–290.

Wirth, K., F.J. Hock, J. Albus, W. Linz, H.G. Alpermann, H. Anagnostopou-los, S. Henke, G. Breipohl, W. Konig, J. Knolle, and B.A. Scholkens(1991) HOE140, a new potent and long-acting bradykinin-antagonist:In vivo studies. Br. J. Pharmacol. 102:774–777.


distribution of bradykinin b2 receptors in sheep brain and spinal cord visualized by in vitro...

Documents