H. Gozlan

Quantitative autoradiography of multiple 5-HT1 receptor subtypes in the brain of control or 5,7-dihydroxytryptamine-treated rats

The distribution of the 2 main types (A and B) of 5-HT1 binding sites in the rat brain was studied by light-microscopic quantitative autoradiography. The 5-HT1A sites were identified using 3H-8-hydroxy-2- (N-dipropylamino)tetralin (3H-8-OH-DPAT) or 3H-5-HT as the ligand. In the latter case, it was shown that 3H-5-HT binding to 5-HT1A sites corresponded to that displaceable by 0.1 microM 8-OH-DPAT or 1 microM spiperone. The “non-5-HT1A” sites labeled by 3H-5-HT in the presence of 0.1 microM 8-OH-DPAT corresponded mainly to 5-HT1B sites. 5-HT1A binding was notably high in limbic regions (dentate gyrus, CA1 and CA3 hippocampal regions, lateral septum, frontal cortex), whereas 5-HT1B binding was particularly concentrated in extrapyramidal areas (caudate nucleus, globus pallidus, substantia nigra). Except in the latter regions, where only one class of 5-HT1 sites was found, both 5-HT1A and 5-HT1B sites existed in all areas examined. The selective degeneration of serotoninergic neurons produced by an intracerebral injection of 5,7- dihydroxytryptamine was associated only with a significant loss of 5- HT1A binding to the dorsal raphe nucleus (-60%) and of 5-HT1B binding to the substantia nigra (-37%). These results are discussed in relation to the possible identity of 5-HT1A and/or 5-HT1B sites with the presynaptic 5-HT autoreceptors controlling nerve impulse flow and neurotransmitter release in serotoninergic neurons.

[3H]8‐Hydroxy‐2‐(Di‐n‐Propylamino)Tetralin Binding to Pre‐ and Postsynaptic 5‐Hydroxytryptamine Sites in Various Regions of the Rat Brain

The specific binding of [3H]8‐hydroxy‐2‐(di‐n‐propylamino)tetralin ([3H]8‐OH‐DPAT) to 5‐hydroxytryptamine (5‐HT)‐related sites was investigated in several regions of the rat brain. Marked differences were observed in the characteristics of binding to membranes from hippocampus, striatum, and cerebral cortex. Hippocampal sites exhibited the highest affinity (KD > 2 nM) followed by the cerebral cortex (KD > 6 nM) and the striatum (KD > 10 nM). Ascorbic acid inhibited specific [3H]8‐OH‐DPAT binding in all three regions but millimolar concentrations of Ca2+, Mg2+, and Mn2+ enhanced specific binding to hippocampal membranes, whereas only Mn2+ increased it in the cerebral cortex and all three cations inhibited specific binding to striatal membranes. Guanine nucleotides (0.1 mM GDP, GTP) inhibited binding to hippocampal and cortical membranes only. As intracerebral 5,7‐dihydroxytryptamine markedly decreased [3H]8‐OH‐DPAT binding sites in the striatum, but not in the hippocampus, the striatal sites appear to be on serotoninergic afferent fibers. In contrast, in the hippocampus the sites appear to be on postsynaptic 5‐HT target cells, as local injection of kainic acid decreased their density. Both types of sites appear to be present in the cerebral cortex. The postsynaptic hippocampal [3H]8‐OH‐DPAT binding sites are probably identical to the 5‐HT1A, subsites, but the relationship between the presynaptic binding sites and the presynaptic autoreceptors controlling 5‐HT release deserves further investigation.

Biochemical evidence for the 5-HT agonist properties of PAT (8-hydroxy-2-(di-n-propylamino)tetralin) in the rat brain

In vitro investigations revealed that PAT (8-hydroxy-2-(n-dipropylamino)tetralin) interacted with postsynaptic 5-HT receptors in the rat brain: the drug stimulated 5-HT-sensitive adenylate cyclase in homogenates of colliculi from new-born rats (KAapp 8.6 microM) and inhibited the specific binding of [3H]5-HT to 5-HT1 sites. The PAT-induced inhibition of [3H]5-HT binding showed marked regional differences compatible with a preferential interaction of PAT (IC50 2 nM) with the 5-HT1A subclass. As previously seen with 5-HT agonists, the efficacy of PAT for displacing [3H]5-HT bound to hippocampal membranes was markedly increased by Mn2+ (1 mM) and reduced by GTP (0.1 mM). PAT also affected presynaptic 5-HT metabolism since it inhibited competitively (Ki 1.4 microM) [3H]5-HT uptake into cortical synaptosomes and reduced (in the presence of the 5-HT uptake inhibitor fluoxetine) the K+-evoked release of [3H]5-HT previously taken up or newly synthesized from [3H]tryptophan in cortical or striatal slices. This latter effect was prevented by 5-HT antagonists (methiothepin, metergoline) suggesting that it was mediated by the stimulation of presynaptic 5-HT autoreceptors by PAT. Like 5-HT, PAT counteracted the stimulatory effect of K+-induced depolarization on the synthesis of [3H]5-HT from [3H]tryptophan in cortical slices. It is concluded that PAT is a potent 5-HT agonist acting on both post- and presynaptic 5-HT receptors in the rat brain.

In Vivo Release of Endogenous Amino Acids from the Rat Striatum: Further Evidence for a Role of Glutamate and Aspartate in Corticostriatal Neurotransmission

By means of the push‐pull cannula method, the outflow of endogenous amino acids was studied in the striatum of halothane‐anesthetized rats. Addition of K + ions (30 mM for 4 min) to the superfusion fluid increased the release of aspartate (+116%), glutamate (+ 217%), taurine (+109%), and γ‐aminobutyric acid (GABA) (−429%) whereas a prolonged decrease in the outflow of glutamine (−28%) and a delayed reduction in the efflux of tyrosine (−25%) were observed. In the absence of Ca2‐, the K+‐induced release of aspartate, glutamate, and GABA was blocked whereas the K + ‐induced release of taurine was still present. Under these conditions, the decrease in glutamine efflux was reduced and that of tyrosine was abolished. Local application of tetrodotoxin (5 μM) decreased only the outflow of glutamate (‐25%). One week following lesion of the ipsilateral sensorimotor cortex the spontaneous outflow of glutamine and of tyrosine was enhanced. Despite the lack of change in their spontaneous outflow, the K +‐evoked release of aspartate and glutamate was less pronounced in lesioned than in control animals, whereas the K + ‐evoked changes in GABA and glutamine efflux were not modified. Our data indicate that the push‐pull cannula method is a reliable approach for the study of the in vivo release of endogenous amino acids. In addition, they provide further evidence for a role for glutamate and aspartate as neuro‐transmitters of corticostriatal neurons.

Physical evidence of the coupling of solubilized 5-HT1A binding sites with G regulatory proteins

Previous investigations (El Mestikawy et al., J Neurochem 51: 1031-1040, 1988) have shown that 5-HT1A binding sites (R[5-HT1A]) solubilized by CHAPS from rat hippocampal membranes can be modulated by guanine nucleotides, as expected from their solubilization together with associated G regulatory proteins (G). Studies of the hydrodynamic properties of solubilized R[5-HT1A] have been presently carried out in order to assess in a more direct way the presence of R[5-HT1A]-G complexes in the soluble extract. Under control conditions, the sedimentation of a CHAPS extract from hippocampal membranes through a 5-30% sucrose gradient (200,000 g, 17 hr, 4 degrees) gave two maxima of [3H]8-OH-DPAT binding activity corresponding to sedimentation coefficients of 8.0 S and 10.0 S, respectively. Running the gradient in the presence of 1 microM GTP revealed a significant reduction of the 10.0 S peak, as expected from the loss of material (probably a G protein) normally associated with R[5-HT1A]. Conversely, attempts to prevent the dissociation of R[5-HT1A]-G by treatment of CHAPS soluble hippocampal extracts with the cross-linking reagent disuccinimidyl suberate (0.1 mM) resulted in a significant increase (+70%) in [3H]8-OH-DPAT binding activity associated with the appearance of a new sedimenting material with a higher coefficient (16.5 S). Furthermore, [3H]8-OH-DPAT binding became almost completely insensitive to guanine nucleotides as expected from the irreversible coupling by disuccinimidyl suberate of R[5-HT1A] with G protein(s). WGA-agarose chromatography of CHAPS soluble hippocampal extract supplemented with GTP allowed the physical separation of R[5-HT1A] from the bulk of G proteins, and a concomitant decrease of [3H]8-OH-DPAT high affinity binding capacity. Partial recovery of the latter could be achieved by reconstituting R[5-HT1A]-G complexes upon the addition of a mixture of pure bovine Gi + Go to G-deprived soluble extracts. Finally in vivo treatment with Pertussis toxin (5 micrograms intracerebroventricularly, 48 hr before killing) resulted in a significant reduction of the specific binding of [3H]8-OH-DPAT (-36%) to hippocampal membranes and corresponding CHAPS soluble extracts, and a marked decrease in the inhibitory effect of GppNHp. Accordingly the G protein associated with R[5-HT1A] belongs probably to the Gi or Go families.

Characteristics of Tyrosine Hydroxylase Activation by K+-Induced Depolarization and/or Forskolin in Rat Striatal Slices

Abstract: The mechanisms of tyrosine hydroxylase (TH) activation by depolarization or exposure of dopaminergic terminals to cyclic AMP have been compared using rat striatal slices. Tissues were incubated with veratridine or 60 mM K+ (depolarizing conditions), on the one hand, and forskolin or dibutyryl cyclic AMP, on the other. K+ ‐(or veratridine‐)induced depolarization triggered an activation of TH (+ 75%) that persisted in soluble extracts of incubated tissues. This effect disappeared when drugs (EGTA, N‐(6‐aminohexyl)‐5‐chloro‐l‐naphthalenesulfon‐amide, Gallopamil) preventing Ca2+‐ and calmodulin‐de‐pendent processes were included in the incubating medium. In contrast, prior in vivo reserpine treatment or in vitro addition of benztropine did not affect the depolarization‐induced activation of TH. In vitro studies of soluble TH extracted from depolarized tissues indicated that activation was associated with a marked increase in the enzyme Vmax but with no change in its apparent affinity for the pteridin cofactor 6‐methyl‐5,6,7,8‐tetrahydrop‐terin (6‐MPH4) or tyrosine. Furthermore, the activated enzyme from depolarized tissues exhibited the same optimal pH (5.8) as native TH extracted from control striatalslices. In contrast, TH activation resulting from tissue incubation in the presence of forskolin or dibutyryl cyclic AMP was associated with a selective increase in the apparent affinity for 6‐MPH4 and a shift in the optimal pH from 5.8 to 7.0–7.2. Clear distinction between the two activating processes was further confirmed by the facts that heparin‐ and cyclic AMP‐dependent phosphorylation stimulated TH activity from K+‐exposed (and control) tissues but not that from striatal slices incubated with forskolin (or dibutyryl cyclic AMP). In contrast, the latter enzyme but not that from depolarized tissues could be activated by Ca2+ ‐dependent phosphorylation. These data strongly support the concept that Ca2+– but not cyclic AMP‐dependent phosphorylation is responsible for TH activation in depolarized dopaminergic terminals.

Synthesis and biological activities of substance P iodinated derivatives

Abstract The synthesis of four substance P (SP) derivatives obtained by coupling the monoiodo and diiodo Bolton and Hunter reagents with synthetic SP is described. These compounds were proved to be good ligands for SP antibodies. As seen for SP, they exhibit a high biological activity in the guinea pig ileum bioassay.

Chromatographic Analyses of the Serotonin 5-HT1A Receptor Solubilized from the Rat Hippocampus

Abstract: Serotonin 5‐HT1A receptors in rat hippocampal membranes were solubilized by 10 mM 3‐[3‐(cholamidopro‐pyl)dimethylammonio]‐l‐propane sulfonate (CHAPS) and chromatographed on various gels in an attempt to design a relevant protocol for their (partial) purification. In particular, an affinity gel made of the 8‐hydroxy‐2‐(di‐n‐propylami‐no)tetralin (8‐OH‐DPAT) derivative 8‐methoxy‐2‐[(N‐propyl, N‐butylamino)amino]tetralin (8‐MeO‐N‐PBAT) coupled to Affigel 202 was specially developed for this purpose. First, studies of the effects of various compounds (detergents, lipids, reducing agents, sugars, etc.) on the specific binding of [3H]8‐OH‐DPAT and on the rate of heat‐induced inactivation of solubilized 5‐HT1A sites led to a buffer composed of 50 mM Tris‐HCl, 50 μM dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 mA/ MnCl2, and 50 μg/ml of cholesleryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5‐HT1A sites, compatible with chromatographic analyses for 2–4 days at 4°C. Adsorption and subsequent elution of [3H]8‐OH‐DPAT specific binding sites were found with several chromatographic gels, including wheat germ agglutinin‐agarose, phenyl‐Sepharose, hydroxylapatite‐Ultrogel, diethyl‐aminoethyl (DEAE)‐Sepharose, and DEAE‐Sephacel. Similarly, 8‐MeO‐N‐PBAT‐Affigel 202 allowed the adsorption and subsequent elution (by 1 mM 5‐HT) of active 5‐HT1Abinding sites solubilized from rat hippocampal membranes. The two‐step chromatography using 8‐MeO‐N‐PBAT‐Affigel 202 followed by wheat germ agglutinin‐agarose gave a fraction enriched (by at least 400‐fold) in 5‐HT1A sites. Sodium do‐decyl sulfate‐polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with Mr close to 60,000.

Irreversible blockade of central 5-HT binding sites by 8-methoxy-2′-chloro-PAT

We have synthesized 8-methoxy-2-(N-2-chloropropyl, N-propyl) aminotetralin (8-methoxy-2-chloro-PAT), an alkylating agent derived from the potent 5-HT agonist, 8-hydroxy-2-(N,N-dipropyl)-aminotetralin (PAT). As expected for an irreversible ligand, the blockade of 3H-PAT or 3H-5-HT binding to post-synaptic 5-HT1 (A and B) sites in rat hippocampal membranes pretreated with 8-methoxy-2-chloro-PAT could not be prevented by extensive washing of membranes. Prior occupancy of 5-HT1 sites by 5-HT or PAT prevented any subsequent irreversible blockade by the alkylating agent. Similar irreversible blockade by 8-methoxy-2-chloro-PAT was found on 3H-PAT binding to striatal membranes suggesting that presynaptic 5-HT binding sites (see Gozlan et al., Nature, Lond. 305, 140, 1983) were sensitive also to the alkylating agent. In contrast, the modifying agent N-ethylmaleimide (NEM) reduced markedly 3H-PAT binding to postsynaptic hippocampal 5-HT1 sites, but did not alter 3H-PAT binding to striatal presynaptic 5-HT sites. Although 8-methoxy-2-chloro-PAT bound irreversibly to different classes of 5-HT binding sites (5-HT1A, 5-HT1B, presynaptic sites), it can be considered a selective alkylating agent, since it exerted no action on 3H-spiperone binding to 5-HT2 sites, 3H-muscimol binding to GABA sites, or 3H-flunitrazepam binding to benzodiazepine sites.

Photo affinity labelling of β-adrenergic receptors of C6 glioma cells: Presence of a nucleophilic group in the receptor☆

Abstract (±) 1-[1-( p -Nitrophenoxy), 2-methyl, 2-propylamino], 3-(α-naphtyloxy), 2-propanol (PNP) was synthesized and found to be a potent photoaffinity label for β-adrenergic receptors of C 6 glioma cells. In the dark, PNP displaced all the [ 3 H]DHA binding sites on C 6 glioma cell membranes ( K D = 5.5 × 10 −8 M). Upon photolysis on isolated C 6 glioma cell membranes: (a) PNP reduced in a dose-dependent manner maximally stimulated β-adrenergic sensitive adenylate cyclase. After extensive washing of the membranes, the maximal β-adrenergic stimulation was reduced without change in the apparent affinity for isoproterenol. A 62% decrease in activity was obtained with 10 −5 M PNP without any change in basal and NaF stimulated adenylate cyclase activities, (b) PNP also irreversibly reduced in a dose-dependent manner the total number of [ 3 H]DHA binding sites without changing the affinity of the remaining sites. The effects of PNP on adenylate cyclase and [ 3 H]DHA binding were suppressed in the presence of (−)alprenolol. Upon photolysis on intact C 6 glioma cells, PNP inactivated β-adrenergic receptors coupled with adenylate cyclase without any change in basal, NaF and Gpp(NH)p stimulated adenylate cyclase activities. These results indicate that PNP photolabelling occurred on the β-adrenergic receptors. Furthermore, as PNP was shown to react with model nucleophiles upon photolysis, this labelling implies the presence of a nucleophilic group in the β-adrenergic receptor.