Maynard H. Makman

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.

Influence of neuroleptic drugs and apomorphine on dopamine-sensitive adenylate cyclase of retina

THE NEUROLEPTIC drugs (major tranquilizers) selectively antagonize a number of behavioral and physiological actions of dopamine, and are believed to exert this effect through a specific postsynaptic blockade of ‘dopamine receptors’ (HORNYKIEWICZ, 1971 ; A N D ~ N et al., 1970; ERNST, 1969; RANDRUP and MUNKVAD, 1970; YEH et al., 1969). Apomorphine mimics the effects of dopamine in several systems and is thought to act by a direct stimulation of the ‘dopamine receptor’ (ANDBN et al., 1967; ERNST, 1969; GOLDBERG, 1968). The effects of the neuroleptic drugs and of apomorphine on the metabolism of dopamine ( A N D ~ N et al., 1967, 1970; NYBACK and SEDVALL, 1968; CHERAMY et al., 1970), as well as their physiological effects on the extrapyramidal system (HAASE and JANSSEN, 1965; D ~ ~ B Y et al., 1971), further suggest that the dopamine receptor can be defined pharmacologically in terms of the actions of these compounds. The biochemical nature of the receptor with which dopamine and these agents interact has not been defined. The effects of catecholamines in a number of peripheral tissues are mediated through activation of adenylate cyclase, and there is evidence indicating that the 13-adrenergic receptor is part of or closely associated with this enzyme system (ROBISON, BUTCHER and SUTHERLAND, 1971). Several lines of evidence suggest that the adenylate cyclase system similarly may mediate the central actions of catecholamines as neurotransmitters (KAKIUCHI and RALL, 1968; SIGGINS et al., 1969; CHASIN eta/., 1971 ; SEEDS and GILMAN, 1971 ; BURKARD, 1972). We have reported previously (BROWN and MAKMAN, 1972) that dopamine, the predominant catecholamine serving as a neurotransmitter in the retina (HAGGENDAL and MALMFORS, 1965; NICHOLS et al., 1967; KRAMER et al., 1971), activates retinal adenylate cyclase and increases the concentration of cyclic AMP in intact isolated retinas. Our finding that dopamine was more potent than epinephrine or norepinephrine as an activator of this enzyme suggested that the receptor associated with the retinal adenylate cyclase was different from the p-adrenergic receptor and indeed might have the pharmacological properties of a specific dopamine receptor. Adenylate cyclase activity was assayed as described (BROWN and MAKMAN, 1972) by the incubation of [a-23P]ATP with homogenates of bovine retina for 5 min at 30°, followed by separation of labelled cyclic AMP by thin layer chromatography and determination of radioactivity. The concentration of cyclic AMP in intact bovine retinas was measured following incubation of the isolated tissue by a modification (BROWN and MAKMAN, 1972; SHERLINE et al., 1972) of the method of GILMAN (1970). We have examined aand 8-adrenergic blocking agents and the neuroleptic drugs, haloperidol, chlorpromazine and fluphenazine, for their capacity to inhibit activation of the adenylate cyclase of calf retina by dopamine. Figure 1 illustrates the inhibition by these agents of the response to 20 p~ dopamine. Propranolol, a potent 8-adrenergic blocking agent, failed to produce greater than a 50% inhibition even at concentrations 25 times that of dopamine. In contrast, haloperidol, chlorpromazine and fluphenazine each inhibited almost completely the activation by equimolar concentrations of dopamine, and caused a 50% blockade when used in concentrations approximately 1/50 that of the catecholamine. Even at equimolar concentrations chlorpromazine sulphoxide, the pharmacologically inactive metabolite of chlorpromazine, did not inhibit the response to 20 p~ dopamine. Haloperidol and the phenothiazines inhibited neither basal adenylate cyclase activity nor activity in the presence of NaF (a nonspecific activator) at the concentrations required to produce complete inhibition of the response to dopamine. Phentolamine and ergotamine also inhibited activation by dopamine of the retinal adenylate cyclase. Phentolamine, an a-adrenergic blocking agent, inhibited at concentrations 50-100 times those necessary for blockade with the neuropletic drugs, and the blockade observed with ergotamine may be unrelated to its a-adrenergic receptor blocking properties since several other ergot alkaloids, none of which are a-adrenergic blocking agents, were equally effective in inhibiting activation by dopamine (OPLER, BROWN and MAKMAN, in preparation). The relative effectiveness of the a-adrenergic blocking agents on the retinal adenylate cyclase is consistent with the influence of these agents in other systems responsive to dopamine. Thus a-adrenergic blocking agents do not block the behavioral and vasodilatory actions of dopamine which are blocked by neuroleptic drugs (ANDBN et al., 1966; MUNKVAD and UDSEN, 1963; YEH et al., 1969), and are less potent than the phenothiazine neuroleptics or than

Striatal met-enkephalin concentration increases following nigrostriatal denervation

Following specific lesion of the nigrostriatal dopaminergic pathways in rat brain, striatal met-enkephalin on the lesioned side increased to 245% of that on the non-lesioned side. This increase was evident only after a lag period of 7 days and the increase was maintained for at least 2 months after lesion. By contrast, there was no change in striatal somatostatin or vasoactive intestinal polypeptide concentration, indicating that the effect was not a generalised one. Levels of all three of these neuropeptides were unchanged in frontal cortex. These findings support the concept of a dopaminergic-enkephalinergic functional interrelationship in the striatum. In addition, the findings provide evidence that, following destruction of nigrostriatal dopaminergic neurons, not only is there a gradually developing postsynaptic dopamine receptor supersensitivity but also a compensatory alteration in the enkephalinergic system.

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.

Morphine receptors in immunocytes and neurons.

Receptor interactions of morphine are reviewed, with particular attention given to a recently discovered opiate receptor, designated mu 3, with unique selectivity for morphine and certain other opiate alkaloids. Morphine, other opiate alkaloids and related analogs are known to bind to the classical delta, mu and kappa opioid receptor subtypes. Each of these subtypes also binds one or more of the endogenous opioid peptides with high affinity. Immunocytes have recently been found to contain a unique receptor for morphine, capable of binding morphine and certain other opiate alkaloids, but with essentially no or exceedingly low affinity for the naturally occurring endogenous opioid peptides or peptide analogs. This putative mu 3 (morphine/opiate alkaloid) receptor is present in invertebrate immunocytes as well as in human peripheral blood monocytes (macrophages). More recently this same receptor has been found in certain established macrophage cell lines and in human peripheral blood granulocytes. Finally, the same or closely related opiate alkaloid-selective (mu 3) receptor has been found to be present in a neuroblastoma and in a hybrid neural cell line. Studies indicate that in the immunocytes the receptor mediates inhibitory effects of morphine on cellular chemotaxis. While the functional coupling of this receptor in neurons is not known, it is postulated that the receptor may mediate effects of opiates on neuronal differentiation and cell division as well as neuronal transmission. Both for the immune system and the nervous system, the mu 3 receptor may constitute a major site of action for putative endogenous morphine or morphine-like substances. This receptor system also provides an additional pharmacological site of action for exogenously administered opiate alkaloid drugs. The mu 3 receptor is proposed to be an important neuro-immune link. This system is likely to play a significant role in a variety of responses involving the immune system, including the response of the organism to stress, infection and malignant transformation.

Occurrence of the opiate alkaloid-selective μ3 receptor in mammalian microglia, astrocytes and Kupffer cells

Evidence is presented for occurrence of opiate alkaloid-selective, opioid-peptide-insensitive receptor binding sites, labeled with [3H]morphine, in primary cultures of cat microglia and cat astrocytes, as well as on highly purified preparations of rat Kupffer cells. These receptors have been designated mu3 on the basis of their close similarity to receptors first found to be present on human peripheral blood monocytes. Exposure of the microglia to morphine and etorphine caused marked quantifiable changes in cellular morphology, including assumption of a more rounded shape and retraction of cytoplasmic processes; in contrast, several opioid peptides were without effect on morphology. The effects of morphine on microglial morphology were blocked by the opiate antagonist naloxone. These effects of drugs on morphology were as predicted for action via the mu3 receptor. Opiate alkaloid binding sites previously detected on the rat C6 glioma cell line were also characterized here as of the mu3 receptor subtype. It is proposed that mu3 receptors have broad distribution in different macrophage cell types of bone marrow lineage, including microglia and Kupffer cells. Furthermore, these receptors are not restricted to cells of bone marrow lineage, since they are also present on astrocytes.

Pertussis toxin blocks depressant effects of opioid, monoaminergic and muscarinic agonists on dorsal-horn network responses in spinal cord-ganglion cultures

After chronic exposure of mouse spinal cord-ganglion explants to morphine, the acute depressant effects of opioids on sensory-evoked dorsal-horn network responses are markedly attenuated, and characteristic cord discharges can then occur even in the presence of greater than 100-fold higher opioid concentrations. The present study demonstrates that a remarkably similar degree of tolerance to opioids develops in these cord-ganglion explants after exposure to pertussis toxin (PTX). The usual acute depressant effects of serotonin, norepinephrine and oxotremorine on dorsal-horn discharges are also similarly attenuated in PTX-treated cultures. PTX is known to interfere with the guanine nucleotide protein Gi that is required for opioid, alpha 2-adrenergic and muscarinic receptor-mediated inhibition of adenylate cyclase in various cells. We have previously found that in cord-dorsal root ganglion explants agents which elevate intracellular cAMP also attenuate opioid depressant effects. Furthermore, these explants contain an opioid-inhibited adenylate cyclase system, and chronic exposure to morphine as well as PTX increases adenylate cyclase activity. These findings together with the present results suggest that the neuromodulatory effects of opioid, monoaminergic and muscarinic agonists on primary afferent networks in the spinal cord may be mediated by binding to neuronal receptor subtypes that are negatively coupled via Gi to a common pool of adenylate cyclase.

Mediation by cyclic AMP of hormone-stimulated glycogenolysis in cultured rat astrocytoma cells

Summary Rat astrocytoma cells, demonstrated to have a hormone-stimulated adenyl cyclase and a cyclic AMP-stimulated protein kinase, degrade glycogen in the presence of norepinephrine, epinephrine, histamine, dibutyryl cyclic AMP, and papaverine, suggesting that cyclic AMP mediates hormonally induced glycogenolysis in astrocytes. Propranolol blocks adrenergic-induced glycogenolysis as well as adrenergic stimulation of adenyl cyclase. Histamine did not activate adenyl cyclase and propranolol did not block histamine-induced glycogenolysis, suggesting that histamine acts in a different manner than do norepinephrine and epinephrine.

Dopamine receptor sensitivity following nigrostriatal lesion in the aged rat.

This investigation sought to determine whether the ability to regulate dopamine receptor sensitivity following removal of dopaminergic innervation is altered during aging. Aged (24-26 months old) Fisher 344 rats compared with young (6 months old) rats had lower levels of dopamine and dopamine receptor binding ([3H]ADTN), but no change of dopamine-stimulated adenylate cyclase activity. Unilateral lesion of the nigrostriatal pathway produced equivalent dopaminergic denervation in rats of both age groups. The denervated striata of young rats had greatly enhanced dopaminergic sensitivity as evidenced by apomorphine induced rotational behavior and increased dopamine stimulated adenylate cyclase activity and [3H]ADTN binding. Old rats responded similarly with a very high degree of increased dopaminergic sensitivity in both the behavioral and biochemical parameters, demonstrating that the ability to regulate dopamine receptors remains basically intact. However, deficits of supersensitivity occurred in apomorphine induced rotational behavior and [3H]ADTN binding and there was a large deficit in the guanine nucleotide sensitive subcomponent of [3H]-ADTN binding. Supersensitivity of dopamine stimulated adenylate cyclase was not altered. The diminished ability to develop supersensitivity to [3H]ADTN binding could contribute to decreased [3H]ADTN binding in unlesioned rats.