Localization of AT(2) receptors in the nucleus of the solitary tract of spontaneously hypertensive and Wistar Kyoto rats using [125I] CGP42112: upregulation of a non-angiotensin II binding site following unilateral nodose ganglionectomy

We have examined the binding distribution of a selective AT(2) receptor ligand [125I] CGP42112 in the brain of adult Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). AT(2) receptor localization was also examined in the rat brainstem following unilateral nodose ganglionectomy. Specific [125I] CGP42112 binding was observed in discrete brain regions from both rat strains, including the nucleus of the solitary tract (NTS), and did not differ between WKY and SHR. [125I] CGP42112 binding in the NTS revealed an AT(2) receptor component that was displaceable by PD 123319 and Ang II (50-58%), as well as a non-angiotensin II receptor component (42-49%). Following unilateral nodose ganglionectomy, [125I] CGP42112 binding density on the denervated side of the NTS was increased approximately two-fold in both WKY and SHR. This increased [125I] CGP42112 binding density in the ipsilateral NTS was comprised of a greater non-angiotensin II component than that observed in the sham groups, since only approximately 30% was displaced by PD123319 and angiotensin II. Furthermore, [125I] CGP42112 also revealed high binding density on the denervated side in the dorsal motor nucleus and the nucleus ambiguus in both WKY and SHR. AT(2) receptor immunoreactivity was also visualised in the NTS of sham operated rats, but was not observed in the dorsal motor nucleus or the nucleus ambiguus, nor was it up-regulated following nodose ganglionectomy. These results demonstrate, for the first time, an AT(2) receptor binding site in the NTS, as well as a non-angiotensin II [125I] CGP42112 binding site. These studies also demonstrate that nodose ganglionectomy represents a useful model in which to study a non-angiotensin II [125I] CGP42112 binding site that is up-regulated following degeneration of afferent vagal nerves.     

Axonal transport of NADPH-diaphorase and [(3)H]nitro-L-arginine binding, but not [(3)H]cGMP binding, by the rat vagus nerve

Previous studies have shown that the NO(ccirf)-cGMP pathway may be functionally relevant in the nodose ganglion and at afferent terminations of the vagus nerve. The technique of unilateral vagal ligations, using double ligatures, was combined with the techniques of NADPH-diaphorase histochemistry, as an index of nitric oxide synthase (NOS) activity, and autoradiography using the radioligands [(3)H]nitro-L-arginine and [(3)H]cGMP, to examine axonal transport of NOS and cGMP-dependent effectors by the rat vagus nerve. A population of perikarya in the nodose ganglia was NADPH-diaphorase positive, and binding of both [(3)H]nitro-L-arginine and [(3)H]cGMP was found on the nodose ganglia. Following vagal ligation, NADPH-diaphorase reactivity accumulated proximal to the proximal ligature and distal to the distal ligature. Vagus nerve transection beyond the distal ligature eliminated NADPH-diaphorase reactivity at the distal ligature. Similarly, [(3)H]nitro-L-arginine binding was found over the nodose ganglion; and after vagal ligation, an accumulation of [(3)H]nitro-L-arginine binding was seen adjacent to the proximal ligature, though little binding was found adjacent to the distal ligature. No accumulation of [3H]cGMP binding was found adjacent to either the proximal or the distal ligatures. These findings suggest that the rat vagus nerve bidirectionally transports NOS, the enzyme involved in biosynthesis of NO(ccirf) by nitroxidergic nerves. As anticipated, [(3)H]nitro-L-arginine, a competitive inhibitor of the amino acid precursor for NO(ccirf), binds only to a centrifugally transported moiety that we conjecture is NOS, while cGMP apparently is not subject to transport. These data further support the use of NO(&z.ccirf;) in transmission at vagal afferent terminals.     

Ontogeny and distribution of opioid receptors in the rat brainstem

The distribution and postnatal ontogeny of opioid receptors have been investigated using in vitro quantitative receptor autoradiography. Rats were studied at postnatal day 1 (P1), P5, P10, P21 and P120 (adult). Opioid receptor sites for (D-Ala2,N-MePhe4,Gly-ol5)-enkephalin (DAMGO) binding were labelled with 4 nM of 3H-DAMGO; (D-Ala2,D-Leu5)-enkephalin (DADLE) binding sites were labelled with 4 nM of 3H-DADLE in the presence of 1 microM unlabelled mu-agonist (N-MePhe3,D-Pro4)-morphiceptin (PL107). We found that both binding sites have strikingly different distributional patterns. [3H]DADLE binding sites were rather homogeneous, whereas the distribution of [3H]DAMGO binding was very heterogeneous with the highest density in the nucleus of the solitary tract (NTS), ambiguus nucleus, dorsal motor nucleus of the vagus and the parabrachial areas. [3H]DAMGO binding density was 2- to 40-fold higher than [3H]DADLE binding sites in most brainstem nuclei. [3H]DAMGO binding sites appeared in most brainstem nuclei at birth, with a high density in cardiorespiratory-related nuclei, whereas [3H]DADLE binding sites were too scarce to be quantitated at P1. Both binding sites increased with age, but the developing patterns depended on the nucleus and the type of binding site. In most areas, the densities of both binding sites reached a maximum between P10 and P21 and then decreased to an adult level, but in some nuclei (e.g. the caudal part of the NTS and dorsal raphe nucleus), [3H]DAMGO binding sites kept increasing until adulthood. In contrast with the brainstem, cortical areas had a lower binding density in the newborn and reached peak levels later than brainstem regions (post P21). We conclude that (1) since [3H]DAMGO binding sites mainly reflect mu-receptors and [3H]DADLE binding sites delta-receptors (in the presence of PL017), the brainstem is essentially a mu-receptor region through delta-receptors are present; (2) both opioid receptors are present at birth but delta-receptors are very scarce in the newborn; (3) both receptors increase with age, but the time course depended on various nuclei and receptor types; (4) cardiorespiratory-related nuclei have high density of mu-receptors at all ages; and (5) opioid receptors develop earlier in the brainstem than in the cortex.     

Association of neurotensin binding sites with sensory and visceromotor components of the vagus nerve

Specific neurotensin (NT) binding sites were recently shown to be highly concentrated in the nucleus of the solitary tract (NTS), which receives primary vagal afferents, and in the dorsal motor nucleus of the vagus (DMN), which contains the cell bodies of origin of vagal preganglionic neurons. To investigate the relationship of these binding sites with sensory and visceromotor components of the vagus nerve, they were labeled here in vitro, using monoiodo[Tyr3]neurotensin (125I-NT) and visualized by light microscopic radioautography in the dorsomedial medulla of both intact and unilaterally vagotomized rats, in the nodose ganglia of intact animals, and in ligated vagus nerves. Unilateral vagotomy performed above the nodose ganglion resulted in a significant ipsilateral decrease in 125I-NT binding within both the NTS and the DMN, suggesting that NT binding sites were associated with both primary afferent fibers and preganglionic nerve cell bodies. The selective radioautographic labeling of a subpopulation (approximately 15%) of neuronal perikarya in the nodose ganglion confirmed that a proportion of vagal afferent neurons contained NT binding sites. Following vagus nerve ligation, a pile up of radiolabeled NT binding sites was observed on both sides of the nerve crush, indicating that NT receptor components were transported both anterogradely and retrogradely along fibers of the vagus nerve. We conclude that NT receptors are synthesized and transported within a subpopulation of afferent and efferent components of the vagus nerve and that NT may therefore act presynaptically upon vagal axon terminals in both central and peripheral nervous systems.     

Binding sites for the glutamate-analogue [H]AMPA in cultured rat brainstem and spinal cord

By means of light microscopic autoradiography, binding sites for the glutamate-analogue [ [³H]AMPA [(RS)-α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid] were observed on the soma and processes of many brainstem and spinal neurons but not on glial cells. Unlabeled AMPA, glutamate or the glutamate antagonist glutamic acid diethyl ester at high concentrations inhibited or reduced binding of [³H]AMPA. Our data indicate that binding sites for [³H]AMPA exhibit characteristics expected for glutamate receptors.     

Biochemical evidence thatl-glutamate is a neurotransmitter of primary vagal afferent nerve fibers

To determine in rat if vagal afferent fibers projecting into the intermediate one third of the nucleus tractus solitarius (NTS), the site of termination of baroafferents, utilize glutamate as a neurotransmitter, the high-affinity uptake of [³H]l-glutamate and content of glutamate were analyzed in micropunches of rat brain stem. The intermediate NTS contains a high-affinity synaptosomal uptake system for [³H]l-glutamate that is greater in capacity than that in areas adjacent to the NTS; it is almost two-fold higher than uptake in medial septum and nucleus accumbens and equal to that of hippocampal regions purportedly containing a rich glutamatergic innervation. Unilateral ablation of the nodose ganglion (i.e. cells of origin of vagal afferents) resulted, within 24 h in a prolonged significant reduction, to 56% of control, of [³H]l-glutamate uptake, bilaterally in the NTS. The reduction of Na⁺-dependent synaptosomal uptake of [³H]l-glutamate, resulted from a decrease in V_max without change in theK_m of the process, was anatomically restricted to the intermediate NTS, and was not associated with changes in [³H]GABA uptake. The content of glutamate in the NTS was significantly (P < 0.01) decreased by 30% 7 days following unilateral extirpation of the nodose ganglion without changes in the concentrations of aspartate, glycine, glutamine, or GABA. A population of vagal afferent fibers projecting to NTS are glutamatergic. The results are consistent with the hypothesis obtained by physiological and pharmacological techniques that glutamate is a neurotransmitter of baroafferents.     

AMPA receptor potentiation by acetylcholinesterase is age-dependently upregulated at synaptogenesis sites of the rat brain

We have used radioligand binding to synaptic membranes from distinct rat brain regions and quantitative autoradiography to investigate the postnatal evolution of acetylcholinesterase (AChE)-evoked up-regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors in CNS areas undergoing synaptogenesis. Incubation of synaptosomal membranes or brain sections with purified AChE caused a developmentally modulated enhancement in the binding of [3H]-(S)-AMPA and the specific AMPA receptor ligand [3H]-(S)-5-fluorowillardiine, but did not modify binding to kainate neither N-methyl-D-aspartate receptors. In all postnatal ages investigated (4, 7, 14, 20, 27, 40 days-old and adult rats), AChE effect on binding was concentration-dependent and blocked by propidium, BW 284c51, diisopropylfluorophosphonate and eserine, therefore requiring indemnity of both peripheral and active sites of the enzyme. AChE-mediated enhancement of [3H]-fluorowillardiine binding was measurable in all major CNS areas, but displayed remarkable anatomical selectivity and developmental regulation. Autoradiograph densitometry exhibited distinct temporal profiles and peaks of treated/control binding ratios for different cortices, cortical layers, and nuclei. Within the parietal, occipital and temporal neocortices, hippocampal CA1 field and cerebellum, AChE-potentiated binding ratios peaked in chronological correspondence with synaptogenesis periods of the respective AMPA-receptor containing targets. This modulation of AMPA receptors by AChE is a molecular mechanism able to transduce localized neural activity into durable modifications of synaptic molecular structure and function. It might also contribute to AChE-mediated neurotoxicity, as postulated in Alzheimer's disease and other CNS disorders.     

Contribution of the vagus nerve to angiotensin II binding sites in the canine medulla

Specific angiotensin II (Ang II) binding sites are present in the dorsal medulla of several species and dose-related cardiovascular effects are produced by microinjection of the peptide into this region. Because the anatomical location of Ang II binding sites in the area postrema (ap), nucleus tractus solitarii (nTS) and dorsal motor nucleus of the vagus (dmnX) coincides with the topography of vagal afferent fibers and efferent motor neurons, the effect of either nodose ganglionectomy or cervical vagotomy on Ang II binding sites in the dorsomedial medulla was investigated in dogs by in vitro receptor autoradiography. Two weeks after unilateral ganglionectomy, there was a marked reduction in the density of specific Ang II binding sites in the ipsilateral ap, nTS and dmnX and an absence of binding sites in the region where vagal afferent fibers course through the rostral medulla. Unilateral cervical vagotomy, which has been shown to spare central processes of afferent fibers, resulted in a loss of binding only in the ipsilateral dmnX. We also show that Ang II binding sites are present in the nodose ganglion and central and peripheral processes of the vagus nerve. The data indicate that medullary Ang II binding sites are associated with both vagal afferent fibers and efferent motor neurons.     

Cholecystokinin and neuropeptide Y receptors on single rabbit vagal afferent ganglion neurons: site of prejunctional modulation of visceral sensory neurons

A [¹²⁵I]cholecystokinin (CCK) analog and [¹²⁵I]peptide YY (PYY) were used to localize and characterize CCK and neuropeptide Y (NPY) receptor binding sites in the rabbit vagal afferent (nodose) ganglion. High concentrations of CCK and NPY binding sites were observed in 10.6% and 9.2% of the nodose ganglion neurons, respectively. Pharmacological experiments using CCK or NPY analogs suggest that both subtypes of CCK (CCK-A and CCK-B) and NPY (Y1 and Y2) receptor binding sites are expressed by discrete populations of neurons in the nodose ganglion. These results suggest sites at which CCK or NPY, released in either the nucleus of the solitary tract or a peripheral tissue, may modulate the release of neurotransmitters from a select population of visceral primary afferent neurons. Possible functions mediated by these receptors include modulation of satiety, opiate analgesia, and the development of morphine tolerance.     

Synaptic interaction of vagal afferents and catecholaminergic neurons in the rat nucleus tractus solitarius

Combined radioautography and immunocytochemistry were used to define the ultrastructure and synaptic relations between vagal sensory afferents and catecholaminergic (CA) neurons of the A2 group located within the nucleus tractus solitarius (NTS) of rat brain. The vagal afferents were radioautographically labeled by tritiated amino acids anterogradely transported from the nodose ganglion. Immunocytochemical labeling for tyrosine hydroxylase (TH) served for the identification of catecholaminergic neurons. The radiographically labeled axons seen by light microscopy were widely distributed throughout the more caudal NTS. The reduced silver grains were more densely distributed within the NTS located homolateral to the injected nodose ganglion. The radioautographically labeled processes were localized in regions containing catecholaminergic neurons as indicated by immunoreactivity for TH. Electron microscopic analysis of the medial NTS at the level of the obex demonstrated that the reduced silver grains were localized within axon terminals. The radioautographically labeled terminals were 2-3 microns in diameter, contained numerous small, clear and a few large, dense vesicles, and formed predominately axodendritic synapses. Many of the recipient dendrites contained immunoreactivity for TH. In rare instances, vagal afferents formed synaptic appositions with both TH-labeled and unlabeled axon terminals and neuronal soma. This study provides the first ultrastructural evidence that the catecholaminergic neurons within the NTS receive direct synapses from sensory neurons in the nodose ganglion.     

Reduced glutamate binding in rat dorsal vagal complex after nodose ganglionectomy

Quantitative receptor autoradiography with L-[3H]glutamate was employed to examine the distribution and properties of glutamate binding sites in the rat brain 14 days after excision of the right nodose ganglion. Slide-mounted coronal sections of the brain showed reduced L-[3H]glutamate binding in the nucleus tractus solitarius/dorsal motor nucleus of the vagus in the ipsilateral relative to the sham-operated side. Densitometric and saturation analyses of binding data indicated a significant reduction in the density of glutamate binding sites (57% decrease relative to sham), while there was a significant increase in receptor affinity (40% greater than sham). Binding was unaltered in the inferior olivary complex. Glutamate receptors are likely to exist on synaptic nerve terminals of vagal afferent fibres within the nucleus tractus solitarius and on vagal preganglionic neurones within the dorsal motor nucleus of the vagus and/or their dendritic processes within the nucleus tractus solitarius. Additionally, our receptor autoradiographic studies provide evidence for L-glutamate being a transmitter of vagal afferent neurones.     

Selective heterologous regulation of 5-HT1A receptor-stimulated 35S GTPgammaS binding in the anterior cingulate cortex as a result of 5-HT2 receptor activation

Previous studies have shown that administration of the 5-HT(2) receptor agonist DOI to rats results in the heterologous desensitization of 5-HT(1A) receptor-mediated behavioral and neuroendocrine responses [Neuropsychopharmacology 19 (1998) 354; J. Neurosci. 21 (2001) 7919]. We hypothesized that the basis for these changes in 5-HT(1A) receptor function may involve changes in the capacity of the 5-HT(1A) receptor to activate G proteins. We examined the effect of chronic administration of DOI on the regulation of 5-HT(1A) receptor function at the level of receptor-G protein interaction using quantitative autoradiography of [(35)S]GTPgammaS binding stimulated by the 5-HT(1A) receptor agonist (+/-)8-OH-DPAT (1 microM). Repeated administration of DOI (1 mg/kg, s.c. once daily for 8 days) resulted in a marked down-regulation in 5-HT(2A) binding sites, as labeled by the antagonist radioligand [(3)H]ketanserin, throughout the cerebral cortex. Chronic DOI treatment also resulted in a significant and selective attenuation of 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding in the anterior cingulate cortex (vehicle-treated: 74+/-7.7% above basal; DOI-treated: 43+/-4.6% above basal). Interestingly, 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding was not altered in the dorsal or median raphe, or in the limbic structures and other cortical regions examined. The decrease in 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding in anterior cingulate cortex was not due to a decrease in 5-HT(1A) receptor number, indicating that the capacity of the 5-HT(1A) receptor to activate G proteins is attenuated in this cortical area following repeated DOI treatment. The heterologous regulation of 5-HT(1A) receptor function by chronic 5-HT(2) receptor activation in the anterior cingulate cortex raises interesting questions as to how the regulatory interaction between these serotonin receptor subtypes influences cognition, memory and emotion.     

Dopamine D2 receptor partial agonists display differential or contrasting characteristics in membrane and cell-based assays of dopamine D2 receptor signaling

Clinical evidence suggests that dopamine D(2) receptor partial agonists must have a sufficiently low intrinsic activity to be effective as antipsychotics. Here, we used dopamine D(2) receptor signaling assays to compare the in vitro functional characteristics of the antipsychotic aripiprazole with other dopamine D(2) receptor partial agonists (7-{3-[4-(2,3-dimethylphenyl)-piperazinyl]propoxy}-2(1H)-quinolinone [OPC-4392], (-)-3-(3-hydroxy-phenyl)-N-n-propylpiperidine [(-)3-PPP] and (+)terguride) and dopamine D(2) receptor antagonists. Aripiprazole and OPC-4392 were inactive in a guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding assay using Chinese Hamster Ovary (CHO) cell membranes expressing cloned human dopamine D(2Long) (hD(2L)) receptors, whereas (-)3-PPP and (+)terguride displayed low intrinsic activity. Aripiprazole also had no effect on [(35)S]GTPgammaS binding to CHO-hD(2L) cells, while OPC-4392, (-)3-PPP and (+)terguride were partial agonists. In contrast, aripiprazole, OPC-4392, (-)3-PPP, and (+)terguride were inactive in a [(35)S]GTPgammaS binding assay using rat striatal membranes. However, at a more downstream level of CHO-hD(2L) cell signalling, these drugs all behaved as dopamine hD(2L) receptor partial agonists, with aripiprazole displaying an intrinsic activity 2 to 3-fold lower (inhibition of forskolin-induced adenosine 3',5'-cyclic monophosphate accumulation) and almost half as high (enhancement of adenosine triphosphate-stimulated [(3)H]arachidonic acid release) as OPC-4392, (-)3-PPP and (+)terguride. Dopamine activity was blocked in each case by (-)raclopride, which was inactive on its own in every assay, as were the antipsychotics haloperidol, olanzapine, ziprasidone and clozapine. Together, these data, whilst preclinical in nature, are consistent with clinical evidence suggesting the favorable antipsychotic profile of aripiprazole, compared with the other clinically ineffective partial agonists, is dependent on its low intrinsic activity at dopamine D(2) receptors. This study also highlights the limitations of using [(35)S]GTPgammaS binding assays to identify dopamine D(2) receptor partial agonists.     

Tachykinin receptor (NK(1), NK(2), NK(3)) binding sites in the rat caudal brainstem following neonatal capsaicin administration

Binding of [125I]-Bolton-Hunter substance P ([125I]-BHSP), [125I]-neurokinin A and [3H]-senktide to tachykinin NK(1), NK(2) and NK(3) receptors, respectively, was examined in caudal brainstem sections of 10-week-old rats pretreated as neonates (P2) with capsaicin (50 mg/kg, s.c.) or vehicle. [125I]-BHSP binding was localised to the nucleus of the solitary tract (NTS), hypoglossal nucleus and inferior olivary complex, whereas [125I]-neurokinin A and [3H]-senktide binding were confined to the NTS. The distribution and density of binding sites were similar in vehicle- and capsaicin-pretreated rats, suggesting that sensory neuron ablation by neonatal capsaicin does not affect tachykinin receptor numbers in the rat caudal brainstem.     

Immunohistochemical detection of glutamate in rat vagal sensory neurons

Vagal primary afferent neurons have their cell bodies located in the nodose (inferior) and jugular (superior) vagal ganglia and send terminals into the nucleus tractus solitarii (NTS) which lies in the dorsomedial medulla. The presence of glutamate (Glu)-containing neurons in the rat nodose ganglion was investigated using immunohistochemistry. Glu-immunoreactivity on nodose sections was found in neuronal perikarya and nerve fibers, but not in non-neuronal elements such as Schwann cells and satellite cells. Both immunoreactive and non-immunoreactive ganglion cells were observed. The immunoreactive ganglion cells amounted to about 60% of the nodose population. No specific intraganglionic localization was observed for the non-immunoreactive cells. Immunoreactive perikarya were slightly smaller than the non-immunoreactive ones, but no relationship was found between size and staining intensities of immunoreactive neurons. The present data indicate that immunodetectable Glu is present in a large population of vagal afferent neurons. They therefore add to a growing body of evidence suggesting that Glu may be the main neurotransmitter released by vagal afferent terminals within the nucleus tractus solitarii.     

The binding of [3H]-5-hydroxytryptamine to homogenates of human brain

Summary The binding of [³H]-5-hydroxytryptamine ([³H]-5-HT) to homogenates of human brain has been studied. The specific binding is saturable, with a K_d (frontal cortex) of 12±2nM, and is inhibited by non-radioactive 5-HT (IC₅₀=26 nM) and D-Lysergic acid diethylamide (IC₅₀=20 nM). Specific, but not non-specific binding of [³H]-5-HT was inhibited by incubation of the homogenates at 50 °C. The binding of [³H]-5-HT across the human brain was not uniform, the highest binding being found in the substantia nigra and hippocampus, and the lowest in the thalamus and pons. The K_d of the binding sites towards 5-HT did, however, appear to be similar for the different brain regions.     

Central and peripheral vagal transport of cholecystokinin binding sites occurs in afferent fibers

The effects of various vagal lesions on cholecystokinin (CCK) binding sites in the nucleus tractus solitarii (NTS) and area postrema (AP) and the peripheral transport of CCK binding sites in the cervical vagus were examined in rats by in vitro autoradiography with [125I]CCK-8. Unilateral supraganglionic, but not subdiaphragmatic vagotomy significantly reduced CCK binding in the ipsilateral NTS. Specific unilateral afferent, but not efferent, vagal rootlet transections also significantly reduced NTS CCK binding ipsilateral to the transections. None of the vagal lesions altered CCK binding in the AP. Infraganglionic but not supraganglionic vagotomy eliminated the peripheral transport of vagal CCK binding sites. Together these results demonstrate that CCK receptors in the NTS are located on vagal afferent terminals, that CCK receptors in the AP are likely postsynaptic to a vagal afferent input and that the peripheral and central transport of vagal CCK binding sites occurs in afferent fibers.     

Choline acetyltransferase activity in the nucleus tractus solitarius: Regulation by the afferent vagus nerve

The influence of nodose ganglionectomy or transection of the peripheral branches of the afferent vagus nerve on choline acetyltransferase (ChAT) activity in the nucleus tractus solitarius (NTS) was studied. ChAT activity was reduced in the microdissected caudal and intermediate portions of the NTS in vagotomized as well as ganglionectomized rats. However, only the ganglionectomy resulted in the degeneration of medullary nerve fibers. These results suggest that the changes in ChAT activity in the NTS are independent of neuronal degeneration and may be due to transynaptic modulation of ChAT activity by afferent vagal impulses. The presence of ChAT in the sensory nodose projection to the NTS, however, cannot be ruled out.     

Dog cerebral cortex contains μ-, δ- and κ-opioid receptors at different densities: apparent lack of evidence for subtypes of the κ-receptor using selective radioligands

The biochemical and pharmacological properties of mu (mu), kappa (kappa) and delta (delta) opioid receptors were ascertained in dog cerebral cortex homogenates. The selective peptides, [3H]D-Pen2-D-Pen5enkephalin [( 3H]DPDPE) and [3H]D-Ala2-MePhe4-Glyol5-enkephalin [3H]Glyol; [3H]DAMGO), bound to delta- and mu-opioid receptors with high affinity (dissociation constants, Kd values = 4.7 and 1.6 nM) but to different densities of binding sites (Bmax values of 49.2 and 6.6 fmol/mg protein, respectively) in washed homogenates of dog cerebral cortex. In contrast, the non-peptides, [3H]U69593 [( 3H]U69) and [3H]etorphine [( 3H]ET), labeled a high concentration of kappa-opioid receptors (respective Bmax values of 67.2 and 76.6 fmol/mg protein) of high affinity (respective Kds of 1.4 and 0.47 nM) in the same tissue homogenates. Thus, the relative rank order of opioid receptor densities was: kappa greater than delta much greater than mu. The selective labeling of the kappa-receptors with two different drugs [( 3H]U69 and [3H]ET) failed to reveal the possible existence of multiple kappa-sites based on the relative Bmax values of the two radioligands. This conclusion was further supported by the similarity of the pharmacological specificity of both [3H]U69 and [3H]ET binding, where all the opioids tested produced 100% inhibition of these labels and where the rank order of potency of opioids at inhibiting the binding of these probes was: U50488 greater than U69593 greater than dynorphin-(1-8) greater than naloxone much greater than morphine much greater than Glyol (DAMGO) greater than DPDPE.(ABSTRACT TRUNCATED AT 250 WORDS)