B. Dvorkin

Distribution of α-adrenergic, β-adrenergic and dopaminergic receptors in discrete hypothalamic areas of rat

Summary Catecholamine receptor binding sites were measured in discrete hypothalamic nuclei or regions as well as in certain extrahypothalamic areas of the adult male rat. For each assay, discrete areas were microdissected from frozen tissue sections and pooled from several animals. Specific high affinity binding sites were assessed at fixed ligand concentrations for [ 3 H] p -aminoclonidine (PAC) and [ 3 H](2-C 2′,6′-(CH 3 O) 2 phenoxyethylamino)-methylbenzodioxan (WB-4101) for α-adrenergic receptor sites, for [ 3 H]dihydroalprenolol (DHA) for β-adrenergic receptor sites, and for [ 3 H]2-amino-6, 7-dihydroxy-1,2,3,4-tetrahydronaphtalene (ADTN) and [ 3 H]spiroperidol in the presence of cinanserin for dopaminergic receptor sites. Regional variations in [ 3 H]WB-4101 binding were relatively small in magnitude, with most hypothalamic and extrahypothalamic areas possessing between 60 and 90% of the binding in frontal cortex. [ 3 H]PAC binding showed a wider range of binding density across brain areas than did [ 3 H]WB-4101, but, in general, variations in [ 3 H]PAC binding paralleled those in [ 3 H]WB-4101 binding. In hypothalamus, binding was characterized as being predominantly to α 1 -receptors in the of [ 3 H]WB-4101 and to α 2 -receptors in the case of [ 3 H]PAC. The medial hypothalamic areas exhibited a somewhat higher density of these α-adrenergic sites than did the lateral hypothalamus (perifornical hypothalamus and medial forebrain bundle). Also, the ratio of [ 3 H]PAC to [ 3 H]WB-4101 binding differed in different hypothalamic areas, ranging from 1.5:1 to 4:1. The median eminence was exceptional in that it contained appreciable [ 3 H]PAC but no significant [ 3 H]WB-4101 binding sites at the radioligand concentrations tested. Binding of [ 3 H]DHA to β-adrenergic receptors varied over approximately a 3-fold range in the different hypothalamic areas, with binding highest in the medial forebrain bundle and the medial preoptic area, and lowest in the periventricular, dorsomedial and posterior hypothalamic nuclei, the median eminence and the zona incerta. The ratio of β-adrenergic to α-adrenergic binding sites was generally lower in the medial than in the lateral hypothalamic areas and higher in the extrahypothalamic areas examined than in the hypothalamus. With regard to [ 3 H]spiroperidol and [ 3 H]ADTN binding to dopaminergic sites, the striatum, nucleus accumbens and olfactory tubercle showed a greater density of [ 3 H]spiroperidol than of [ 3 H]ADTN sites, in contrast to the hypothalamus where [ 3 H]ADTN binding was more predominant. Within the hypothalamus, [ 3 H]ADTN binding was relatively uniform, while [ 3 H]spiroperidol binding was quite high in four hypothalamic areas (lateral perifornical area, medial forebrain bundle, paraventricular and dorsomedial nuclei), intermediate in the median eminence and arcuate nucleus, and low or not detectable in all other hypothalamic areas.

Evidence for loss of brain [3H]spiroperidol and [3H]ADTN binding sites in rabbit brain with aging

[3H]Spiroperidol and [3H]2-amino-6,7-dihydroxyl-1,2,3,4,-tetrahydronaphthalene hydrochloride (ADTN) binding were measured in various central nervous system regions of 5 month and 5.5 year old rabbits. In striatum, young animals had a 38% higher number of [3H]spiroperidol binding sites and a 140% higher number of [3H]ADTN binding sites than did the older animals. In frontal cortex and anterior limbic cortex there were respectively 42% and 26% more [3H]spiroperidol binding sites in the young animals. There was no change in the binding site number or affinity for [3H]spiroperidol in retina with aging. Pharmacological characterization demonstrated that [3H]spiroperidol binds to a dopamine receptor in striatum and to a serotonin receptor in cortex.

Modulation of adenylate cyclase activity of mouse spinal cord-ganglion explants by opioids, serotonin and pertussis toxin

Organotypic cultures of fetal mouse spinal cord-ganglion explants (2-4 weeks in vitro) contain forskolin-stimulated adenylate cyclase (AC) activity that is inhibited by levorphanol and other opioid agonists in a dose-dependent manner. Inhibition by levorphanol no longer occurs if sodium is omitted from the incubation and the levorphanol inhibition is blocked by the opioid antagonist, naloxone. These findings together with the ineffectiveness of dextrorphan indicate that the opioid inhibition of forskolin-stimulated AC is receptor mediated. Both the delta- and kappa-receptor subtypes appear to be involved since the selective delta-opioid agonist, [D-Pen2, D-Pen5]enkephalin, and the selective kappa-opioid agonist, t-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)cyclohexyl]-benzene acetamide (U-50,488H) are both effective at nanomolar concentrations. In contrast, the selective mu-opioid agonist, Tyr-D-Ala-Gly-N-MePhe-Gly-ol, has no significant effect even at micromolar concentrations. Both cord and ganglion components of the explants contain opioid-sensitive AC. Forskolin-stimulated AC of the explants is also inhibited by serotonin and carbachol. The serotonin effect appears to be mediated by 5-HT1A receptors, based on relative agonist and antagonist selectivity. Chronic exposure of cultures to morphine results in enhanced basal and forskolin-stimulated AC as well as attenuation of opioid-inhibition of AC assayed in the presence of forskolin; treatment of explants with pertussis toxin causes similar changes in the AC system. The inhibitory effect of serotonin is also attenuated by the pertussis toxin treatment. Basal AC activity of the explants (assayed without forskolin present) is stimulated to a small but significant extent by opioids and by serotonin. The opioid stimulatory effect is markedly enhanced following either morphine or pertussis toxin treatment of the explants. The attenuation of opioid- and serotonin-inhibition of AC produced by chronic exposure to pertussis toxin and the attenuation of opioid inhibition produced by exposure to morphine are consonant with the attenuation of opioid and monoaminergic depression of sensory evoked dorsal horn network responses after similar chronic treatments. It is proposed that the inhibitory effects of opioids and serotonin on these neurons are mediated by receptors that are negatively coupled via a pertussis toxin sensitive Gi protein to AC. Furthermore, alterations of AC with chronic morphine treatment may be involved in the development of physiologic tolerance to opioids.

Properties of dopamine agonist and antagonist binding sites in mammalian retina.

Retinal homogenates of calf, rat, rabbit and Cebus appella and Macaca mulata monkeys were found to contain stereospecific binding sites for the dopamine antagonist [3H]spiroperidol. In further studies with calf and rat retina, stereospecific binding sites were also found for the dopamine agonist [3H]ADTN (2-amino-6,7,-dihydroxy-1,2,3,4-tetrahydronapththalene). The [3H]spiroperidol binding sites in calf retina were pharmacologically similar to the dopaminergic spiroperidol binding sites previously demonstrated to be present in striatum. However, calf and rabbit retina contained less than 1/10 the concentration of [3H]spiroperidol binding sites found in striatum. Saturation studies and Scatchard analyses showed a single class [3H]spiroperidol binding sites with Kd (apparent dissociation constant) = 0.3 and 0.2 nM and Bmax (binding site number) = 38 and 24 fmol/mg protein in calf retina and rabbit retina respectively. Rates of [3H]spiroperidol association and dissociation were also evaluated in calf retina. Drug specificity for [3H]ADTN binding in calf retina resembled that previously reported for striatal [3H]ADTN binding and thus differed from retinal [3H]spiroperidol binding. Calf retinal [3H]ADTN binding sites had a Kd = 9 nM and Bmax = 113 +/- 12 fmol/mg protein. Thus, the total number of [3H]ADTN sites in retina was at least twice that of [3H]spiroperidol sites. Guanine nucleotides (GTP and Gpp (NH)p) but not ATP reduced the affinity of the dopamine agonist ADTN for [3H]spiroperidol binding, and also reduced the specific binding of [3H]ADTN itself up to a maximal value of about 50% of control binding. Saturation studies of calf retinal [3H]ADTN binding confirmed that Gpp(NH)p-displaceable sites were a discrete saturable subset of stereospecific [3H]ADTN sites with Kd = 9 nM and Bmax = 50 +/- 6 fmol/mg protein. The Gpp(NH)p insensitive sites had a Kd = 9 nM and Bmax = 63 +/- 7 fmol/mg protein. It is proposed that although [3H]ADTN sites differ pharmacologically from [3H]spiroperidol sites, since [3H]spiroperidol sites are guanine nucleotide-sensitive and similar in number to the guanine nucleotide-sensitive class of [3H]ADTN sites, they may possibly be related to these sites as well as to adenylate cyclase. In addition, retina contains guanine nucleotide-insenstive [3H]ADTN sites, possibly presynaptic and probably not coupled to adenylate cyclase.

Murine macrophage cell lines contain μ3-opiate receptors

Opiate alkaloid-selective, opioid peptide-insensitive mu 3 receptors are present in three murine macrophage cell lines (J774.2; RAW 264.7; BAC1.2F5). The receptor binds morphine, its active metabolite morphine 6-glucuronide and certain other alkaloids, but not morphine 3-glucuronide or any of the opioid peptides tested. The cell lines thus provide valuable model systems for investigation of mu 3-opiate receptors, previously demonstrated to mediate inhibitory effects of morphine on activation of human peripheral blood macrophages (monocytes).

Presence and characterization of nociceptin (orphanin FQ) receptor binding in adult rat and human fetal hypothalamus

High affinity and saturable nociceptin (orphanin FQ) receptors were detected and characterized in adult rat and human fetal hypothalamic membranes, utilizing [125I]Tyr12-nociceptin as ligand. Nociceptin bound with picomolar affinity, dynorphin A with nanomolar affinity, naloxone and dynorphan A(1-8) with micromolar while des-Tyr1-dynorphin (dynorphin A(2-17)), several other opioids, morphine and benzomorphans failed to compete for binding at 1-10 microM. Gpp(NH)p together with sodium ion markedly decreased binding, consistent with involvement of a G protein-linked receptor.

Binding sites for [3H]SCH 23390 in retina: properties and possible relationship to dopamine D1-receptors mediating stimulation of adenylate cyclase

Bovine, rat and chick embryo retinal membranes contain high affinity, saturable and stereospecific binding sites for the selective dopamine D1-receptor antagonist, [3H]SCH 23390 (R-(+)-8-chloro-2,3,4,5-tetrahydro-3- methyl-5-phenyl-lH-3-benzazepin-7-ol). Saturation studies and Scatchard analyses showed a single class of [3H]SCH 23390 binding sites with Kd (apparent dissociation constant) values of 0.5-1.4 nM for the different species studied. A high ratio of specific to non-specific binding was found over a wide range of radioligand concentrations. The Bmax (binding site number) for [3H]SCH 23390 in calf retina was 307 +/- 38 fmol/mg protein, significantly greater than Bmax values previously obtained for binding of [3H]spiroperidol and [3H]ADTN (2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene) to dopamine D2-receptors in calf retina. Relative affinities (Ki values) of dopamine antagonists for calf retinal [3H]SCH 23390 binding sites were similar to those reported for [3H]SCH 23390 binding in rat striatum and also were in general agreement with potencies for antagonism of retinal dopamine-stimulated adenylate cyclase. However, they differed markedly from the relative affinities for retinal D2-receptor sites. Additional data indicated that SCH 23390 did not bind significantly to retinal D2- or serotonergic receptors and had 30- to 80-fold less affinity for alpha 2-noradrenergic than for the [3H]SCH 23390 sites. Competition studies indicated a high degree of selectivity for dopamine agonist over other agonists for [3H]SCH 23390 binding sites with Ki values in the range expected for interaction with dopamine receptors mediating stimulation of adenylate cyclase. Affinity for dopamine was decreased in the presence of the GTP analogue, Gpp(NH)p. In the presence of sodium ions the affinities of dopamine agonists for [3H]SCH 23390 binding sites were markedly and selectively decreased; the sensitivity to dopamine for stimulation of adenylate cyclase activity was also decreased in the presence of sodium ions. Modulation by sodium ions was previously observed for D2- but not for a D1-receptor interaction. It is proposed that [3H]SCH 23390 binds to a unique class of receptors, most likely D1-receptors coupled to adenylate cyclase in retina. [3H]SCH 23390 provides a potent new tool for study of these receptors. In retina D1-receptors positively coupled to cyclase as well as D2- and other receptors that may be negatively coupled to cyclase, appear to be regulated by sodium ions as well as by guanine nucleotides.

3H-spiroperidol binding and dopamine-stimulated adenylate cyclase: Evidence for multiple classes of receptors in primate brain regions

Abstract Studies of displacement by agonist and antagonist drugs of 3H-spiroperidol binding in brain regions of Cebus and rhesus monkeys revealed one type of receptor in caudate nucleus and a second type of receptor in both frontal and anterior limbic cortex. Compared with caudate, the cortical regions were more sensitive to clozapine and loxapine, equally sensitive to fluphenazine and relatively less sensitive to haloperidol. Also, the cortical regions were insensitive to molindone. Parallel studies using the dopamine-stimulated adenylate cyclase have demonstrated three types of receptors, one in caudate, a second in frontal cortex, and a third in anterior limbic cortex. In each region studied, relative sensitivities to drug using these two methods differed, suggesting that in each of these regions only a relatively small portion of 3H-spiroperidol receptors are coupled to adenylate cyclase.

Catecholamine-stimulated cyclic AMP formation in phenylethanolamine n-methyltransferase containing brain stem nuclei of normal rats and of rats with spontaneous genetic hypertension

Stimultaion of cyclic AMP formation by epinephrine and norepinephrine has been studied in discrete areas of rat brain that include the epinephrine-containing brain stem nuclei C-1 and C-2. In the C-1 area, epinephrine-stimulated cyclic AMP formation was partially reversed by 100 microM phentolamine and by 10--100 microM propranolol or alprenolol and hence appeared to involve activation of a mixture of both alpha- and beta-adrenergic receptors as has been reported for other rat brain areas such as the cerebral cortex. However, in the C-2-area, the epinephrine and norepinephrine stimulated cyclic AMP formation involved the activation of a single receptor type which was alpha-like in character. Stimulation of cyclic AMP formation by epinephrine in the C-2 area was antagonized by nanomolar concentrations of both phentolamine and yohimbine. The epinephrine-stimulated formation of cyclic AMP in the C-2 but not in the C-1 area was augmented in a strains of rats which exhibit spontaneous genetic hypertension (SHR) vs. Wistar-Kyoto controls. It is suggested that the enhanced epinephrine-stimulated cyclic AMP formation in the C-2 area of SHR rats could be a physiological compensatory response to some other hypertension-causing lesion which, for example, results in chronically reduced epinephrine release or in ruduced availability of epinephrine at its postsynaptic receptor thereby leading to receptor supersensitivity. Supporting this possibility was the finding that treatment of SHRs and control animals and reserpine resulted in enhancement of epinephrine-stimulated cyclic AMP formation in the C-2 area of control rats, essentially obliterating the difference between control and SHR. The findings are also interepreted as supporting the involvement of epinephrine neurons in central vaso-depressor mechanisms.

Presence in neuroblastoma cells of a μ3 receptor with selectivity for opiate alkaloids but without affinity for opioid peptides

Evidence is presented for the occurrence of a unique opiate alkaloid-selective, opioid peptide-insensitive binding site in N18TG2 mouse neuroblastoma cells and in late passage hybrid F-11 cells, derived from N18TG2 neuroblastoma cells and rat dorsal root ganglion cells. Those cells lacked classical opioid peptide-sensitive receptor subtypes, but contained [3H]morphine and [3H]diprenorphine binding sites with affinity for certain opiate alkaloids but not for any endogenously occurring opioid peptide or peptide analog tested, including D-ala2-D-leu5-enkephalin (DADLE), D-Ala2,N-Me-Phe4,Gly5-ol (DAGO) and dynorphin A(1-17). The binding site differed from hitherto described mu, delta and kappa neuronal opioid receptors not only on the basis of peptide insensitivity, but also on the basis of selectivity and affinities of alkaloids. Saturation experiments with [3H]morphine indicated the presence of a single site with Kd = 49 nM and Bmax = 1510 fmol/mg protein. This novel binding site was not present in F-11 hybrid cells at early passage. Instead the hybrid cells contained conventional opioid receptors (predominantly delta and also mu) capable of binding DADLE and other peptides as well as opiate alkaloids. With additional passage (cell divisions) of the hybrid cells, during which a limited change occurred in mouse chromosome number, the peptide-insensitive binding appeared and the opioid peptide-binding (delta and mu) receptors were lost reciprocally. Thus, expression of the peptide-insensitive binding normally may be repressed when conventional opioid receptors are expressed. The peptide-insensitive opiate binding site described here appears to correspond to the mu 3 receptor subtype, recently identified pharmacologically and functionally in several cell types of the immune system.(ABSTRACT TRUNCATED AT 250 WORDS)