Richard Brière

Specific [3H]SCH23390 Binding to Dopamine D1 Receptors in Cerebral Cortex and Neostriatum: Evidence for Heterogeneities in Affinity States and Cortical Distribution

The tritiated antagonist SCH23390 was used to identify dopamine D1 receptors in the cerebral cortex and neostriatum. The kinetic properties of binding were investigated in parallel experiments with membrane preparations from both tissues. The densities of receptors (Bmax) and the dissociation constants (KD) were determined from saturation curves, and the specificity of binding verified in competition experiments using agonists and antagonists. The cortical D1 receptor displays the same pharmacological selectivity (including stereospecificity) and kinetic properties as the neostriatal D1 receptor. From both the dissociation kinetics by dilution and the competition curves, it could be established that there is an heterogeneity of binding, probably due to high‐ and low‐affinity states. Endogenous dopamine, 4‐hydroxy‐3‐methoxyphenylacetic acid, 3,4‐dihydroxyphenylacetic acid, and 3‐methoxytyramine contents, as well as D1 receptor distribution, were measured for the neostriatum and four localized cortical areas: anterior cingulate. primary somatosensory, primary visual, and piriform‐entorhinal. For the regions examined, the distribution of D1 receptors is heterogeneous, but correlates very well (r > 0.98) with the endogenous levels of dopamine and its major metabolites.

Effects ofp-chlorophenylalanine on cortical monoamines and on the activity of noradrenergic neurons

The catecholamines noradrenaline, dopamine, adrenaline, the indoleamine 5-hydroxy-tryptamine (5-HT; serotonin), and some of their major metabolites were assayed, using high performance liquid chromatography (HPLC), in the neocortex of normal rats as well as in animals in which 5-HT synthesis had been inhibited withp-chlorophenylalanine. Besides important depletions in serotonin and in 5-hydroxyindole-3-acetic acid, noradrenaline levels were significantly reduced, but the content in 3-methoxy-4-hydroxyphenylglycol was increased, indicating an augmented utilization of this amine. The levels of dopamine and 3-methoxytyramine were also reduced, although homovanillic acid and 3,4-dihydroxyphenylacetic acid levels remained constant. The spontaneous unitary activity of identified noradrenergic neurons in the Locus coeruleus was increased, indicating an hyperactivity of this system. These results can be interpreted in relation to functional interactions between the catecholamines and serotonin; i.e.: a decrease in endogenous serotonin results in the loss of a negative feedback control of noradrenaline release.

Adrenergic receptor and catecholamine distribution in rat cerebral cortex: binding studies with [3H]prazosin, [3H]idazoxan and [3H]dihydroalprenolol☆

The tritiated adrenergic antagonists [3H]dihydroalprenolol ([3H]DHA; beta-receptors), [3H]prazosin ([3H]PRZ; alpha 1-receptors), and [3H]idazoxan ([3H]IDA; alpha 2-receptors) were used to determine the distribution of these sites in 5 defined areas of the adult rat cerebral cortex. The highest density of [3H]PRZ binding was found in the prefrontal cortex, with a lower and homogeneous distribution for the frontal, parietal, occipital and temporal areas. The [3H]IDA binding sites were fairly uniform for all areas, except for the temporal cortex where it was very dense. In contrast, beta-adrenoceptors labelled by [3H]DHA were very homogeneous for all the regions examined. The functional significance of the distribution of alpha 1, alpha 2 and beta-adrenoceptors is discussed in relation to the catecholamine innervation and monoamine contents measured by high performance liquid chromatography.

Long-term unilateral noradrenergic denervation: Monoamine content and 3H-prazosin binding sites in rat neocortex☆

Direct biochemical determinations of alpha 1 adrenoceptor sites were performed in the neocortex of rats subjected to a selective unilateral noradrenergic deafferentation, obtained by microinjecting 6-OHDA in the right dorsal noradrenergic bundle (DNB). After 3 months survival the alpha 1 sites were assayed using 3H-Prazosin (3H-PRZ), in both the denervated and the contralateral (control) cortex. The catecholamines dopamine (DA), epinephrine (EPI), norepinephrine (NE), as well as the indoleamine serotonin (5-HT) were measured using radioenzymatic assays in samples of the frontal cortex of these same animals, as well as in the septum and hippocampus in order to assess the extent and specificity of the deafferentation. The results document that unilateral NE-deafferentations of the cerebral cortex are feasable, the reduction in NE persists for at least three months, and there is an increased endogenous DA content. In the denervated cerebral cortex specific binding of 3H-PRZ showed an increase (+ 45%) in the density of receptor sites (Bmax) without any changes in the dissociation constant (KD 25 degrees C). The results have to be considered in relation both to plasticity changes in monoamine fibers and to denervation-induced alterations of the postysynaptic alpha-adrenoceptors.

Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: Role of disulfide and sulfhydryl groups

The triated adrenergic antagonists Prazosin ([3H]PRZ) and Idazoxan ([3H]IDA, or RX-781094) bind specifically and with high affinity to α1 and α2-adrenoceptors respectively, in membrane preparations from cerebral cortex. Saturation experiments performed to determine the density of receptors and the dissociation constant (Kd) were analyzed by the methods of Eadie Hofstee, iterative modelling, and the procedure of Hill, while the specificity of the labelling was verified by displacement experiments. Since receptors are proteins, we examined the role of disulfide (−SS−) bridges and sulfhydryl (−SH) groups in the specific combination of [3H]PRZ and [3H]IDA to the α1 and α2-adrenoceptors. Pretreatment of the membranes with the −SS− reactive DL-dithiothreitol (DTT) or the alkylating agent N-ethylmaleimide (NEM), alone or in combination, decreased specific binding of both ligands, with only minor changes in the non-specific counts. The [3H]IDA binding (α2-sites) was more sensitive to both DTT and NEM than the [3H]PRZ sites (α2-adrenoceptors), and the initial changes induced by alkylation of the α2-site were due to an important decrease in the affinity for [3H]IDA, as judged by the increase in theKd. This modulation in the affinity caused by alkylation of a thiol group could explain the higher potency of the blocking agent tetramine disulfide benextramine at the α2-site. The results provide evidence for the participation of −SS− and −SH groups in the binding site of α1 and α2-adrenoceptors in the cerebral cortex.

Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: competition studies with [3H]prazosin and [3H]idazoxan

The tritiated adrenergic antagonists prazosin ([3H]PRZ) and idazoxan ([3H]IDA, or RX-781094) bind specifically and with high affinity in membrane preparations from cerebral cortex to alpha-1- and alpha-2-adrenoceptors respectively. Saturation experiments, performed to determine the density of receptors (Bmax; maximum binding capacity) and the dissociation constant (Kd 25 °C), were analyzed by the methods of Eadie and Hofstee, iterative modelling, and the procedure of Hill. The pharmacologic properties and specificity of the labelling was verified by displacement experiments using alpha-adrenergic antagonists and agonists. The antagonist drugs showed the following order of potency to displace [3H]prazosin: prazosin ≫ phentolamine ≫ corynanthine > pyrextramine ≫ yohimbine ≫ piperoxan > benextramine > idazoxan; for the agonists: clonidine ≫ (−)-noradrenaline ≫ (−)-adrenaline ≫ phenylephrine, while other drugs, such as (−)-propranolol, dopamine, (−)-isoproterenol and serotonin only competed with the alpha-1-ligand at concentrations above 20 μM. The alpha2-sites labelled by [3H]idazoxan were characterized by the antagonist displacement sequence idazoxan ≫ phentolamine > yohimbine = > piperoxan ≫ pyrextramine ≫ benextramine ≫ prazosin ≫ corynanthine. The agonists order of potency to compete with [3H]idazoxan was clonidine ≫ phenylephrine = > (−)-adrenaline > (−)-noradrenaline, and for other related drugs it was (−)-propranolol ≫ dopamine ≫ serotonin > (−)-isoproterenol. These competition experiments clearly showed two pharmacologically distinct sites, but question the relative specificity of some of the adrenergic drugs.

Selective noradrenergic denervation and 3H-prazosin binding sites in rat neocortex

Biochemical determinations of alpha 1-noradrenergic binding sites were performed in the neocortex of normal rats and of rats which had been subjected to a selective noradrenergic deafferentation obtained by microinjecting 6-OHDA in the dorsal noradrenergic bundle. The extent and the specificity of the deafferentation was assessed by measuring the catecholamines dopamine, epinephrine and norepinephrine, as well as the indoleamine serotonin using radioenzymatic assays. In the denervated animals (90% reduction in NE levels) specific binding of the alpha 1-noradrenergic antagonist prazosin revealed an increase only in the number of binding sites (Bmax) without changes in the dissociation constant (Kd).

Evidence for the participation of disulfide and sulfhydril groups in the specific binding of [3H]prazosin in cerebral cortex

The tritiated α1 antagonist prazosin [3H]PRZ binds specifically and with high affinity to postsynaptic adrenoceptors in membrane preparations from cerebral cortex. Since adrenoceptors are of protein nature, it was of interest of investigate the possible role of disulfide (—SS—) and sulfhydril (—SH) groups in the binding of [3H]PRZ. Pretreatment of the membranes with the disulfide and sulfhydryl reactivesdl0Dithiothreitol,l-Dithiothreitol, Dithioerythritol or 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB), alone or in combination with the alkylating agent N-Methylmaleimide (NMM), decreased specific [3HPRZ binding, with minor changes in the non-specific counts. Saturation experiments revealed that all these reagents reduced the affinity of the binding site for [3H]PRZ, as judged by theKd 25°C, but only the alkylating agent NMM and the oxydizing reagent DTNB produced in addition to the increase inKd, a decrease of the maximum binding capacity (Bmax). The present results provide evidence for a participation of—SS—and/or—SH groups in the recognition site of the α1-adrenoceptor of cerebral cortex.

Dopamine in the Neocortex: A Classical Monoamine Neurotransmitter as a Trace Amine

The mammalian cerebral cortex receives catecholamine-containing afferents, and at least two such systems have been very well documented: [1 the noradrenergic (NA) projections originating from the Locus coeruleus or A6 region, and 2] the cortical dopaminergic (DA) fibers arising from the Substantia nigra and the adjacent ventral tegmental area, or A9 and A10 regions (Dahlstrom and Fuxe, 1964; Ungerstedt, 1971). The presence of significant numbers of catecholamine-containing axonal varicosities, presumed to be the terminals and release sites, was first demonstrated by fluorescence histochemistry. Based on these anatomical findings it was soon implied that they could be acting as conventional neurotransmitters in cerebral cortex. This was supported by the in vivo release studies of cortical DA and NA (Reader et al., 1976), as well as by numerous electrophysiological investigations (Bunney and Aghajanian, 1976; Krnjevic and Phillis, 1963; Reader, 1978, 1980; Reader et al., 1979a; Stone, 1973) showing effects of these CA upon cortical neurons.

The Heterogeneity of the Catecholamine Innervation of Cerebral Cortex

The central catecholamine (CA) neurons in the brain stem and their CNS projections were the first to be described in correlative biochemical and histofluorescent investigations as chemically identified neurotransmitter systems. By combining histofluorescent, autoradiographic, and immunocytochemical methods with biochemical determinations of endogenous levels and activities of the enzymes of synthesis, three types of CA neuronal systems were described in the CNS, according to the monoamine they can synthesize and presumably use as their neurotransmitter, i.e., dopamine (DA; Berger et al, 1974; Hokfelt et al, 1974; Lindvall et al, 1974), noradrenaline (NA; Carlsson et al., 1962; Dahlstrom and Fuxe, 1964; Fuxe, 1965; Fuxe et al., 1968; Ungerstedt, 1971; Lindvall and Bjorklund, 1974), and adrenaline (AD; Hokfelt et al, 1973; Van der Gugten et al, 1976; Versteeg et al, 1976; Reader, 1981).