Proc. Natl. Acad. Sci. USAVol. 85, pp. 40614065, June 1988Neurobiology

Characterization and visualization of rat and guinea pig brain KCopioid receptors: Evidence for icK and K2 opioid receptors

(opioid receptor subtypes/quantitative autoradiography/comparative neurochemistry/K opioid analgesics/dynorphin)

R. SUZANNE ZUKIN, MAHBOUBEH EGHBALI, DIANE OLIVE, ELLEN M. UNTERWALD, AND ANN TEMPEL

Departments of Neuroscience and Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461

Communicated by Michael V. L. Bennett, January 5, 1988

ABSTRACT K opioid receptors (K receptors) have beencharacterized in homogenates of guinea pig and rat brain underin vitro binding conditions. K receptors were labeled by usingthe tritiated prototypic K opioid ethylketocyclazocine underconditions in which ,u and 8 opioid binding was suppressed. Inthe case of guinea pig brain membranes, a single population ofhigh-affinity K opioid receptor sites (K sites; Kd = 0.66 nM,BMMX = 80 fmol/mg of protein) was observed. In contrast, inthe case ofrat brain, two populations ofK sites were observed-high-affinity sites at low density (Kd = 1.0 nM, Bm. = 16fmol/mg of protein) and low-affinity sites at high density (Kd =13 nM, Bin" = 111 fmol/mg of protein). To test the hypothesisthat the high- and low-affinity K sites represent two distinct Kreceptor subtypes, a series of opioids were tested for theirabilities to compete for binding to the two sites. U-69,593 andCambridge 20 selectively displaced the high-affinity K site inboth guinea pig and rat tissue, but were inactive at the rat-brainlow-affinity site. Other K opioid drugs, including U-50,488,ethylketocyclazocine, bremazocine, cyclazocine, and dynor-phin (1-17), competed for binding to both sites, but withdifferent rank orders of potency. Quantitative light microscopyin vitro autoradiography was used to visualize the neuroana-tomical pattern of K receptors in rat and guinea pig brain. Thedistribution patterns of the two K receptor subtypes of rat brainwere clearly different. The pattern of rat high-affmiity K sitesparalleled that of guinea pig in the caudate-putamen, mid-brain, central gray substance of cerebrum, and substantianigra; interspecies differences were apparent throughout mostof the rest of the brain. Collectively, these data provide directevidence for the presence of two K receptor subtypes; theU-69,593-sensitive, high-affinity K, site predominates in guineapig brain, and the U-69,593-insensitive, low-affinity K2 sitepredominates in rat brain.

Opiates and opioid peptides produce their pharmacologicalactions by interactions with the 4, 8, K, and a- opioidreceptors (,u, 8, K, and a- receptors; for a review, see ref. 1).Pharmacological studies have established that ketocyclazo-cine-like opioids produce their antinociceptive and uniquesedative actions through an interaction with K receptors (2).These drugs effect a more pronounced sedation than do otheropioids and have been evaluated as anesthetic agents. Kopioid drugs neither suppress morphine abstinence nor in-duce abstinence in morphine-dependent monkeys (3). Theendogenous opioid peptide dynorphin also interacts with highselectivity at K receptors.Evidence for a separate K receptor distinct from the

morphine (A) and enkephalin (Enk; 8) receptors has beenprovided by pharmacological (2, 4), electrophysiological (5,6), binding (4, 7, 8), and solubilization and purification (9-11)studies. In vitro autoradiography was used to visualize K

receptors in rat (12) and guinea pig brain (13); guinea pig Kreceptors were shown to be selectively localized in the deeplayers of the neocortex. Rat brain K receptors were notcharacterized biochemically. Because most neurophysio-logical, behavioral, and biochemical studies of the endoge-nous opioid systems have been undertaken in rat, we havecarried out a study of the properties and anatomical distri-bution of K receptors in these two species. The present studyprovides direct evidence for the presence of two K receptorsubtypes: the high-affinity K1 receptor site (K1 site) predom-inates in guinea pig brain, and the low-affinity K2 sitepredominates in rat brain.

MATERIALS AND METHODSBinding Assay. Rats (male, Sprague-Dawley, 150-200 g) or

guinea pigs (male, Hartley, 300-400 g) were decapitated, andbrains were immediately removed. Brain tissue preparationand binding assays were carried out as described (14).Aliquots (1 ml; 0.8-0.9 mg/ ml of protein) offreshly preparedhomogenate from whole brain were incubated in triplicatewith tritiated ethylketocyclazocine ([3H]EKC; 0.1-25 nM) in50 mM Tris HCl buffer (pH 7.4) at 40C for 60 min in theabsence or presence of 10 AM nonradioactive EKC. Forblockade of A and 8 receptors, the binding assay was carriedout in the presence of 100 nM [D-Ala2,MePhe4,Glyol5]Enk inwhich MePhe is N-methylphenylalanine and Glyol is glycinoland 100 nM [D-Ala2,D-Leu5]Enk. [3H]Bremazocine was notused in these studies because it labeled an additional site inbrain not labeled by [3H]EKC and not displaceable by ,u, 8,K, or or opioids. Scatchard data were analyzed by computer-assisted linear regression analysis (in the case of monophasiccurves) and by the program LIGAND (15) (in the case ofbiphasic curves). Each experiment was carried out a mini-mum of three times. Competition analyses were carried outwith 2 nM [3H]EKC in guinea pig brain assays to ensurespecific binding to K1 receptors (Kd = 0.7 nM) or 10 nM[3H]EKC in the presence of 1 ,uM U-69,593 for rat brainassays to ensure specific binding to K2 sites (Kd = 13 nM).

Autoradiography. Autoradiographic studies were carriedout with tritium-sensitive film as described (16). Male Sprague-Dawley rats and male Hartley guinea pigs were sacrificed bydecapitation; brains were rapidly removed, and coronalsections (20 ,um) were prepared. Brain sections were incu-bated for 1 hr at 4°C in 50 mM Tris-HCl buffer (pH 7.4) witheither 10 nM [3H]EKC in the presence of 100 nM [D-Ala2,MePhe4,Glyol5]Enk and 100 nM [D-Ala2,D-Leu5]Enk (tolabel total K receptors) or in 10 nM [3H]U-69,593 {tritiated(5a,7a,8,8)-( - )-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro-(4,5)dec-8-yl]benzeneacetamide} (to label K1 receptors). Ad-jacent sections were incubated under the same conditions ina solution containing radiolabeled ligand in the presence of

Abbreviations: [3H]EKC, [3H]ethylketocyclazocine; Glyol, glyci-nol; Enk, enkephalin; ,u, 8, K, and a-receptor(s), IL, 8, K, and oaopioidreceptor(s); K sites, K receptor sites.

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1000-fold excess (10 ,uM) of unlabeled levorphanol or U-69,593 to assess nonspecific binding. Nonspecific binding inevery case was <8 + 2% of total binding. Adjacent brainsections were stained with cresyl violet for histologicalverification of structures, and the anatomical structures ofinterest were identified by reference to the rat atlas ofPaxinos and Watson (17). The optical density of each struc-ture was determined, and receptor densities were computedas described in the legend to Table 3.

Drugs. [3H]EKC (45.95 Ci/mmol; 1 Ci = 37 GBq) wasprovided by the National Institute on Drug Abuse. [D-Ala2,MePhe4,Glyol5]Enk and haloperidol were purchasedfrom Sigma; [3H]U-69,593 (42.1 Ci/mmol) from New En-gland Nuclear; and [D-Ala2,D-Leu5]Enk and dynorphin(1-17), from Peninsula Laboratories (San Carlos, CA). EKCand cyclazocine were given by Sterling-Winthrop (Rensse-laer, NY); U-50,488H {trans-(+)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamidemethanesul-fonate} and U-69,593, by Upjohn; Cambridge 20, by J.Hughes ofParke, Davis; tifluadom, by Anaquest (Murry Hill,NJ) and bremazocine, by H. B. A. Welle of ACF Chemie-farma (The Netherlands).

RESULTSPharmacological Characterization of K1 and ac2 Opioid

Binding Sites. Scatchard analysis of specific [3H]EKC bind-ing (unblocked) to guinea pig brain membranes revealed abiphasic curve suggesting binding to be at least two classes ofbinding sites (Fig. 1). Computer-assisted nonlinear regression

L._1%10

0

00LZ

[3H] Ethylketocyclazocine Bound (fmol/mg protein)

FIG. 1. Scatchard analyses of [3H]EKC binding to guinea pigbrain membranes. *, Total specific binding, defined as the totalbinding of [3H]EKC minus binding in the presence of 10 ,uMnonlabeled EKC; o, specific binding to K receptors, defined asbinding of [3H]EKC in the presence of 100 nM [D-Ala2,Me-Phe4,Glyol5]Enk (,u receptor blocker) and 100 nM [D-Ala2,D-Leu5]Enk (8 receptor blocker) minus binding in the presence of 10AM nonlabeled EKC and the ,u and 8 blockers at 100 nM; A, specificbinding to K2 receptors, defined as binding of [3H]EKC to Kreceptors, except that 5 ,uM U-69,593 was included in all samples;and A, binding of [3H]EKC in the presence of 100 nM [D-Ala2,Me-Phe4,Glyol5]Enk, 100 nM [D-Ala2,MePhe4,Glyol5]Enk, and 5 AMU-50,488h minus binding in the presence of 10 ,uM EKC and allblockers. Data are from one experiment, performed in triplicate,which was repeated two times. Data as shown were analyzed by acomputer program for nonlinear least-squares regression analysis.

analysis (15) of the data afforded a best fit for a curvecalculated for two binding components. The first was char-acterized by a Kd = 0.38 + 0.1 nM and a B = 86 ± 8fmol/mg of protein; and the second, by a Kd = 21 ± 9 nMand a Bmax =349 ± 27 fmol/mg of protein. Biphasic curveswere also observed for unblocked [3H]EKC binding to ratbrain membranes (Fig. 2). The density of the lower affinitybinding component computed from the Scatchard plot wassubstantially greater in guinea pig than in rat brain (Bmax 2 =349 vs. 173 fmol/mg of protein).K receptors were specifically labeled in preparations of rat

and guinea pig brain membranes by using the K opioid[3H]EKC in the presence of saturating concentrations (100nM) of nonlabeled [D-Ala2,MePhe4,Glyol5]Enk (JL ligand)and [D-Ala2,D-Leu5]Enk (S ligand) to block binding of theradioligand to ju and 8 receptors (1, 7, 12). In the case ofguinea pig membranes (Fig. 1), a linear Scatchard plot wasobserved-(Kd - 0.66 ± 0.09 nM and Bm - 80 + 7 fmol/mgof protein). In contrast, in the case of rat brain membraneswith At and 8 receptor ligands (Fig. 2), the Scatchard plotremained biphasic (Kdl = 1.0 nM, Bm.1 = 16 fmol/mg ofprotein; Kd2 = 13.1 nM, Bmx2 = 111 fmol/mg of protein).Inclusion of U-69,593 (5 jLM), a specific K receptor ligand(Fig. 2), or Cambridge 20 (data not shown) selectivelyeliminated the higher affinity component of the Scatchardplot obtained for rat brain membranes with Au and 8 receptorblockers; in contrast, U-50,488 displaced [3H]EKC bindingfrom both sites. Increasing the concentration of ,u and 8receptor blockers to 1 ,uM resulted in data indistinguishablefrom that obtained at a concentration of 100 nM. Table 1summarizes K1 and K2 receptor affinities and densities ob-tained for the two species by Scatchard analysis. Together,

m

0

U-

c

co

P-L

100 200 300[3H] Ethylketocyclazocine Bound (fmol /mg protein)

FIG. 2. Scatchard analyses of [3H]EKC binding to rat brainmembranes. *, Total specific binding; o, specific binding to Kreceptors; *, specific binding to K2 receptors; and A, binding of[3H]EKC in the presence of 100nM [D-Ala2,MePhe4,Glyol]Enk, 100nM [D-Ala2,D-Leu5]Enk, and 5 ,uM U-50,488h. Receptor definitionsand data analysis were as described in the legend to Fig. 1. Data arethe means from a representative experiment, which was repeatedtwo times.

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Table 1. Binding parameters for [3H]EKC binding to rat andguinea pig brain K receptors

Parameters for K receptors

K1 K2

Kd, Bmax, fmol/mg Kd, Bmax, fmol/mgBrain nM of protein nM of proteinRat 1.0 ± 0.5 16 ± 8 13 ± 4 111 ± 16Guinea

pig 0.66 ± 0.1 80 ± 7 ND NDWhole rat or guinea pig brain minus cerebellum was prepared and

binding was carried out as described. Values reported are the means± SEM of three independent experiments. ND, not detected.

these results document the presence of high-affinity K1receptors in guinea pig brain and both high- and low-affinityK2 sites, the latter predominating, in rat brain.To further characterize the different K receptor subtypes in

rat and guinea pig brain membranes, a series of drugs weretested for their abilities to compete for K receptor binding inthe two tissues (Table 2). The rank order ofpotency observedfor K2 receptors in rat brain [dynorphin > bremazocine >>(-)EKC > cyclazocine >> U-50,488h; U-69,593 was inac-tive] is clearly different from that observed for K1 receptorsin guinea pig brain [(- )EKC > bremazocine > U-50,488h >cyclazocine > U-69,593]. In particular, U-50,488h was muchmore potent at the guinea pig K1 site than at the rat brain K2site, and U-69,593 was specific for the K1 receptor subtypethat predominates in guinea pig brain. That both sites arelikely to be relevant K receptors and not nonspecific sites issuggested by their selectivity for K opioids (Table 2) and theirsensitivity to heat and protease treatment (data not shown).Competition analyses showed that U-69,593 displaced onlyabout 85% of K receptor binding in guinea pig membranes(data not shown). The residual binding could result from asmall population of K2 receptors. In contrast, U69,593 dis-placed only 15% of K receptor binding in rat brain tissue (datanot shown). Moreover, nonlabeled EKC competed for thebinding of [3H]EKC to rat brain membranes with an Kj of 44nM when the radioligand concentration was 10 nM (favoringbinding to the low-affinity site) and with an Kj of0.5 nM whenthe radioligand was 1 nM (favoring binding to the high-affinitysite) (data not shown). A comparison of the drug potency

A E

C

Table 2. Relative potencies of opioids in inhibiting [3H]EKCbinding to rat and guinea pig brain K receptors

Parameters for K receptors

Guinea pig brain K1 Rat brain K2

Relative RelativeDrug Ki, nM potency* Ki, nM potency*

(-)-EKC 0.7 ± 0.15 1 44 ± 5t 1Dynorphin 6.7 ± 8.0t 0.1 1.7 ± 1.2 26Bremazocine 0.9 0.8 3.9 ± 0.4 11Tifluadom 2.7 ± 0.40 0.26 39 ± 26 1.1Cyclazocine 4.3 0.16 65 ± 20 0.68Cambridge 20 5.1 ± 3.40 0.14 151 ± 27 0.29U-50,488h 2.4 ± 2.0t 0.29 484 ± 110 0.09U-69,593 20.8 ± 9.6 0.03 >10,000 <0.01Normorphine >10,000 <0.0001 >10,000 <0.01[D-Pen2 5]Enk >10,000 <0.0001 >10,000 <0.01Haloperidol >10,000 <0.0001 >10,000 <0.01TCP >10,000 <0.0001 >10,000 <0.01Membranes ofwhole rat or guinea pig brain minus cerebellum were

prepared and binding was carried out as described. Each determi-nation represents the mean ± SEM of three independent experi-ments. TCP, N-[1-(2-thienyl)cyclohexyl] piperidine; Pen, penicilla-mine.*Potencies relative to (- )-EKC in the same tissue.tCalculated from the Ki determined for (±)-EKC, assuming that(+ )-EKC is inactive.tDeterminations represent the means ± SD of two independentexperiments.

profiles for the predominant receptor in the two speciesrevealed a good fit to a two-site model (correlations r = 0.88and r = 0.94 for the two sites; data not shown). Collectively,these data suggest that there are two distinct K binding sites,a U-69,593-sensitive high-affinity K1 receptor that predomi-nates over a small fraction of low-affinity sites in guinea pigbrain and a U-69,593-insensitive low-affinity K2 receptor thatpredominates over a small fraction of high-affinity sites in ratbrain.

Neuroanatomical Distribution of Rat and Guinea Pig KReceptors. To visualize neuroanatomical patterns of K recep-tors, quantitative light-microscopy autoradiography was car-ried out on thaw-mounted sections of frozen rat and guineapig brain (Fig. 3-5 and Table 3). At the level of the anterior

w.t.t

FIG. 3. Autoradiograms of selectedcoronal guinea pig brain sections labeledwith [3H]EKC in the presence of 100 nM[D-Ala2,MePhe4,Glyol5]Enk and 100 nM[D-Ala2,D-Leu5]Enk to visualize K1 re-ceptors. FrPaSS, frontoparietal cortex,somatosensory area; CP, caudate-pu-tamen; cc, corpus callosum; FrPa, fron-toparietal cortex; Hi, hippocampus; CM,central medial nucleus of the thalamus;VP, ventroposterior nucleus of the thal-amus; BL, basolateral nucleus of theamygdala; SuG, superficial gray layer ofthe superior colliculus; CG, central gray;SN, substantia nigra reticulata; Cb, cer-ebellum; LC, locus coeruleus.

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A B

Table 3. Regional distribution of K opioid receptor densities insections of rat and guinea pig brain as determined byquantitative autoradiography

K opioid receptor densities

Guinea pig Rat brainbrain K1, K1 + K2,fmol of fmol of K1, fmol of[3H]EKC [3H]EKC [3H]U-69,593per mg of per mg of per mg of

Region tissue* tissue* tissue

C D

FIG. 4. Autoradiograms of selected coronal rat brainlabeled with [3H]EKC in the presence of 100 nM [D-Ala2,1Glyol5]Enk and 100 nM [D-Ala2,D-Leu5]Enk to label K1receptors. For abbreviations, see Fig. 3.

commissure (Fig. 3A), K1 receptors of guinea pi4selectively localized at a moderate density in the surof the striatum and were absent in the striasomes. GuiK1 receptors were also of moderate density in the bed r

ofthe stria terminalis. Rat brain K1 sites (Fig. 5A) had psimilar to that of guinea pig K1 sites in the caudate-purat K2 sites (total minus K1) occurred at a particulardensity in the bed nucleus of the stria terminalismoderate density in the striasomes.

K1 receptors of guinea pig were selectively localize(deep layers V and VI of the neocortex (Fig. 3 A and BK receptors of rat were nearly uniform throughout theof the neocortex with the exception of layer III, whewere not detected (Fig. 4A and B); boundaries correspto the known cytoarchitectural layers were not visicontrast, rat K1 receptors (Fig. 5 A and B) were ofdensity in layers IV-VI.At the level of the guinea pig diencephalon (Fig.

sites occurred at high density in the molecular layer

Hii-

CM

.. ..

BA

NeocortexLayers I and IILayer IIILayer IVLayers V and VI

StriatumPatchesSurrounds

Nucleus accumbenssections Bed nucleus of the striaMePhe4, terminalisand K2 Claustrum

Endopiriform nucleusFundus striati

g were HippocampusTrounds Molecular layerinea pig Pyramidal cell layernucleus Granular cell layeratterns Thalamic nuclei (in total)ltamen; Paraventricular nucleusPly high Laterodorsal nucleusand at Posterior nuclear group

Centrolateral nucleus1 in the Intermediodorsal). Total nucleusD layers Rhomboid nucleusre they Gelatinosus

ionding Medial geniculateible. In nucleushigher Hypothalamic nuclei

Dorsal hypothalamic3B), K1 aer of the Ventromedial nucleus

Zona incertaAmygdaloid nucleiSubstantia nigra

FrPa reticulataInterpeduncular nucleusSuperficial gray layer of

superior colliculus. BL Cerebellum

Central gray substanceLocus coeruleusSpinal tract of trigeminalnerve

Corpus callosum

25.5 ± 0.525.5 ± 0.525.5 ± 0.554.6 ± 2.7

30.9 ± 0.930.9 ± 0.949.1 ± 1.9

61.9 ± 4.361.3 ± 1.9

NDND

56.4 ± 3.452.8 ± 3.2

ND7.3 ± 0.1tNDNDNDND

41.9 ± 2.1ND

41.9 ± 2.141.9 ± 2.1

71.0 ± 2.345.5 ± 2.358.2 ± 3.5

81.9 ± 7.487.8 ± 8.2

NDND

28.6 ± 4.341.9 ± 1.751.0 ± 2.6

ND61.3 ± 1.472.8 ± 2.983.7 ± 3.4

ND 98.3 ± 4.9ND 92.8 ± 9.3ND 56.4 ± 0.2

10.3 ± 2.110.3 ± 2.114.3 + 2.49.2 ± 1.8

ND17.5 + 2.030.9 ± 1.8

18.8 ± 1.625.5 + 2.424.7 ± 2.142.0 + 1.6

ND

21.2 ± 1.6NDND

10.6 ± 0.8

12.5 ± 0.1NDND

34.6 ± 1.0 ND 9.7 ± 1.8

9.1 ± 0.19.1 ± 0.1NDND

51.0 ± 1.056.4 ± 3.453.3 ± 2.392.3 ± 8.8

24.1 ±19.3 ±22.6 ±18.5 ±

58.2 ± 1.2 51.0 ± 1.3 14.3 ±63.7 ± 1.9 72.8 ± 2.2 14.8 ±

76.4 ± 3.156.4 ± 3.445.5 ± 0.947.3 ± 5.2

78.3 ± 3.132.8 ± 1.056.4 ± 1.178.3 ± 8.6

2.22.01.91.5

2.22.8

20.7 ± 3.0ND

19.8 ± 2.0ND

29.1 ± 0.9 43.7 ± 1.8 ND3.6 ± 0.0 7.3 ± 0.1 ND

C D

FIG. 5. Autoradiograms of selected coronal sections from ratbrain labeled with [3H]U-69,593 to visualize K1 receptors. Forabbreviations, see Fig. 3.

Sections of rat or guinea pig brain were labeled in vitro withradioligands specific for K1 or total K receptors. Corrected densito-metric readings (in optical density units) were averaged and con-verted to receptor density values (fmol/mg) by reference to astandard curve for brain tissue, computed by using tritium standards(Amersham). Receptor density values are reported as means ± SEMof averaged values from the corresponding sections of three rats orguinea pigs after correction for the contributions caused by nonspe-cific binding and to background film density. ND, not detected.*[3H]EKC binding was carried out in the presence of 100 nM[D-Ala2,MePhe4,GlyolI]Enk and 100 nM [D-Ala2,D-Leu5]Enk.tGuinea pig brain exhibited low and uniform density of K receptorsin the thalamic nuclei.

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dentate gyrus and in the pyramidal cell layer of the hippo-campal formation. In contrast, rat K1 receptors were absentfrom the hippocampus, while K2 receptors were of highdensity in the pyramidal cell layer as well as the CA1, CA2,and CA3 areas (of the hippocampus); and the granular layerof the dentate gyrus (Fig. 4B).

In the thalamic and hypothalamic areas, striking differ-ences were seen between the two species and between thetwo receptor subtypes. In the case of guinea pig brain (Fig.3B), K1 receptors were selectively localized in the medialgeniculate nucleus of the thalamus. K1 receptors of rat wereundetectable in the thalamus except in the paraventricularnucleus and the medial geniculate and (at low levels) in thecentrolateral and intermediodorsal nuclei (Fig. 5B). K2 recep-tors occurred in the posterior nuclear group and the medialand midline nuclear groups, including the centrolateral nu-cleus, the intermediodorsal nucleus, and the rhomboid andgelatinosus nuclei (Fig. 4B). Both K1 and K2 receptors of ratwere of moderate density in the dorsal hypothalamus andventromedial nucleus of the hypothalamus.At the level of the midbrain, guinea pig K1 receptors were

at high density in the central gray area, the substantia nigra,the interpeduncular nucleus, and the superior colliculus (Fig.3C). Rat K2 receptors (Fig. 4C) paralleled guinea pig K1receptors at this level; high levels occurred in the periaque-ductual gray area (primarily the dorsal area), the substantianigra reticulata, and the interpeduncular nucleus. Particu-larly striking was the high density of K receptors in thesuperficial gray layer of the superior colliculus. Rat K1receptors exhibited a distribution similar to that of total Ksites but were lower in density by a factor of =5 (Fig. SC). Atthe level of the brainstem (Fig. 3D), guinea pig K1 receptorswere at high density in the cerebellum and central gray of thepons and at moderate density in the locus coeruleus, and thespinal tract of the trigeminal nerve. Rat K2 receptors were ofhigh density in the cerebellum, the locus coeruleus, and thecentral gray area of the pons (Fig. 4D) and of moderatedensity in the spinal tract of the trigeminal nerve (fifth nerve).Rat K1 receptors were not detectable in the locus coeruleus,cerebellum, or spinal tract of the trigeminal nerve (Fig. SD).

DISCUSSIONThe present study shows, on the basis of both in vitro bindingassays and quantitative receptor autoradiography at the levelof the light microscope, the presence of K receptor sites inboth guinea pig and rat brain. An important differencebetween the two species is the presence of a population ofhigh-affinity K sites in guinea pig brain that is found at lowdensity in rat brain and the presence at a high density of alower affinity site in rat brain that is at low density (or absent)in guinea pig brain. Several findings indicate that the K sitesthat predominate in the two species represent different Kreceptor subtypes. First, the two sites have different affinities(for the K1 subtype, Kd = 1 nM; for the K2 subtype, Kd = 13nM). Second, the two sites exhibit different ligand selectivityprofiles. In particular, U-50,488h and Cambridge 20 are muchmore potent at the guinea pig (K1) site than at the predominantrat brain (K2) site, and U-69,593 is specific for K1 (inactive atthe K2 site). Third, the two receptor sites exhibit differentneuroanatomical distribution patterns, as evidenced by au-toradiography on sections of rat brain.What might be the functional significance oftwo K receptor

subtypes in rat brain? In analogy to M1 and M2 muscarinicreceptors, it is possible that K1 and K2 opioid receptors coupledirectly to different channel types or couple to the same ordifferent channel types through different second-messengersystems. A recent electrophysiological study by Shen andCrain showed that at nanomolar concentrations, dynorphinand U-50,488h produced prolongation of the action potential

duration in some mouse dorsal root ganglion neurons andshortening in others (18). Our finding of K receptor subtypesis consistent with this observation.Our finding of two K receptor subtypes in rat brain is not

surprising in view of the recent finding of at least fourfunctional muscarinic receptor genes in this tissue (19). More-over, several other laboratories have reported evidence for Kreceptor subtypes. For example, Castanas et al. (20) reportedthe presence of K1, K2, and K3 binding sites in adrenal medullaon the basis of multiphasic binding isotherms. Iyengar et al.(21) provided physiological evidence for K receptor heteroge-neity by determining differential sensitivity of K opioids toWIN 44,441-3 antagonism in two neuroendocrine systems- (i)the hypothalamic-pituitary-adrenal axis and (it) release ofplasma thyroid-stimulating hormone. Our study extends theseprevious studies by identifying ligands specific or highlyselective for each of the two K receptor subtypes and byidentifying tissues highly enriched in each site.Our autoradiographic studies clearly indicate that K1 and K2

receptors have different distributions in rat brain. Thesestudies also show that, although the K sites of rat and guineapig brain exhibit similar distributions in the midbrain andhindbrain, they exhibit strikingly different patterns in severalforebrain structures such as the striatum, nucleus accumbens,neocortex, and hippocampal formation. The most strikingdifferences between the species and subtypes are seen in thethalamic and hypothalamic areas. The differences in neuro-anatomical distribution support the finding from our receptorbinding studies of heterogeneity in the K receptor system.

This work was supported by National Institutes of Health GrantsDA 04439 (to R.S.Z.) and NS 21973 (to A.T.). R.S.Z. is the recipientof a Research Career Development Award DA 00069 from theNational Institute on Drug Abuse. E.M.U. is a postdoctoral traineefunded by National Institutes of Health Training Grant MH 1578.

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