Micheline Vial

Characterization and autoradiographic distribution of vasoactive intestinal peptide binding sites in the rat central nervous system.

Biochemical characteristics and topographical distribution of mono-[125I )vasoactive intestinal peptide (VIP) binding sites in rat brain were studied on tissue sections and by quantitative autoradiography. Biochemical investigations show two classes of binding sites with a dissociation constant of 1.03 +/- 0.11 nM and 68 +/- 14 nM and a maximal binding capacity of 43.3 +/- 5.1 fmol/mg protein and 713 +/- 117 fmol/mg protein respectively. The order of potency of various peptides to inhibit 125I-VIP binding to brain sections is: VIP greater than PHI greater than secretin greater than VIP greater than hGRF. Autoradiography reveals the highest densities of binding sites in the pineal gland, the dentate gyrus of the hippocampus, the central amygdaloid nucleus and in various thalamic nuclei such as the mediodorsal, lateral posterior, submedius, dorsolateral and medial geniculate nuclei. Similar high densities are observed in the olfactory bulbs as well as in the suprachiasmatic and dorsomedial nuclei of the hypothalamus and in the superior colliculus. These data together with the distribution of the endogenous peptide suggest a physiological role for VIP both in the regulation of CNS activities and pituitary functions.

Characterization and autoradiographic distribution of neurotensin binding sites in the human brain

The characteristics and topographical distribution of monoiodo 125I-Tyr3-neurotensin (NT) binding sites in normal human brain tissue were studied on brain sections and by quantitative autoradiography. Sections at the level of the substantia nigra show a dissociation constant and maximal binding capacity of 4.8 +/- 0.8 nM and 70 +/- 7 fmol/mg protein, respectively. High density of 125I-NT binding sites were mainly found in dopaminergic (DA)-rich areas such as the substantia nigra, the ventral tegmental area, the striatum and the nucleus accumbens, further supporting an interaction between NT and DA neurons in human brain.

Quantitative autoradiography of the rat brain vesicular monoamine transporter using the binding of [3H]dihydrotetrabenazine and 7-amino-8-[125I]iodoketanserin

The binding of [3H]dihydrotetrabenazine, a specific ligand of the monoamine transporter present on serotonin and catecholamine synaptic vesicles, was studied on rat brain sections. The characteristics of binding (Kd = 5.0 nM, k1 = 0.13 x 10(6) M-1 s-1; k-1 = 0.66 x 10(-3) s-1) were similar to those previously observed on tissue homogenates. The rostrocaudal topographical distribution of dihydrotetrabenazine binding sites was analysed by quantitative autoradiography. High labelling was observed in regions richly innervated by monoaminergic systems: dopamine in the striatum and olfactory tubercles, noradrenaline in the striatal fissure and in the paraventricular and dorsomedial hypothalamus and serotonin in the lateral septum, islands of Calleja and suprachiasmatic nucleus. Cell bodies were also labelled in the substantia nigra and ventral tegmental area (dopamine), in locus coeruleus (noradrenaline) and in raphe nucleus (serotonin). The pituitary gland (particularly the neural lobe) and the pineal gland were also labelled. Low labelling was observed in various areas of the cerebral cortex and in the cerebellum. Unilateral 6-hydroxydopamine lesion of the substantia nigra dramatically reduced [3H]dihydrotetrabenazine labelling in the ipsilateral striatum. Moreover, ketanserin has recently been shown to possess a nanomolar affinity for the vesicular monoamine transporter, and autoradiographic localization of brain monoaminergic synaptic vesicles was also obtained by means of the derivative 7-amino-8-[125I]iodoketanserin in the presence of 5-hydroxytryptamine2 and alpha 1 antagonists, although the non-specific labelling was higher than with [3H]dihydrotetrabenazine. It is concluded that [3H]dihydrotetrabenazine may represent a valuable monoaminergic marker in in vitro autoradiographic studies.

In vitro biochemical characterization and autoradiographic distribution of 3H-thyrotropin-releasing hormone binding sites in rat brain sections.

In the present study, we describe the biochemical characteristics and the autoradiographic distribution of thyrotropin-releasing hormone (TRH) receptors in the rat central nervous system (CNS) after in vitro incubation of brain slices with 3H-TRH. Scatchard analysis showed that, in the range of concentrations tested (0.7-35 nM), 3H-TRH bound to a single-class of receptors with a dissociation constant of 6 nM and a number of binding sites of 20 fmol/mg protein. Increasing concentrations of unlabeled TRH produced a dose-dependent inhibition of 3H-TRH binding. The only analogue as potent as TRH to displace 3H-TRH binding was 3-Me-TRH, whereas 1-Me-TRH or TRH-free acid as well as pGlu-His, pGlu-Pro-NH2 or His-Pro-diketopiperazine were ineffective. Neither Luteinizing hormone-releasing hormone (LHRH), neurotensin, somatostatin, D-Ala-Met-enkephalin nor VIP showed any significant affinity for TRH binding sites. Autoradiograms obtained by apposition of LKB 3H-Ultrofilm showed that the highest concentrations of 3H-TRH binding sites were found in the ventral dentate gyrus of the hippocampal formation, the lateral amygdaloid nucleus, the nucleus accumbens, and the thalamic paraventricular nucleus. The biochemical characterization of 3H-TRH binding in brain sections is in good agreement with previous reports on membrane preparations and the autoradiographic localization of the binding sites provides anatomical support for the effects of TRH in the CNS.

Corticosteroid receptors in rat hippocampal sections: Effect of adrenalectomy and corticosterone replacement

Rat brain sections, located at the hippocampal level, were used to study the effect of bilateral adrenalectomy, with or without corticosterone treatment, on the number and affinity of corticosteroid binding sites. Adrenalectomy induces an increase of corticosterone receptor binding sites whereas adrenalectomy followed by in vivo corticosterone treatment produces a 50% decrease of binding site number. Increases and decreases of binding site number were not associated with a significant modification of the affinity for corticosterone. The present data show that in vivo corticosterone modulates its own number of binding sites demonstrated by in vitro binding on brain sections, in a manner which is reminiscent of changes in cytosol receptors demonstrated by conventional biochemical methods. Thus, this in vitro method provides an alternative way to study the plasticity of hippocampal glucocorticoid receptors.

The effect of vasoactive intestinal peptide (VIP) on the contractile activity of human uterine smooth muscle.

1. In the present study we examined the in vitro effect of vasoactive intestinal peptide (VIP) on spontaneous contractions in both inner and outer layers of non‐pregnant human myometrium. A dose‐dependent relaxation was observed, but with a marked difference in sensitivity to VIP between the two layers, with an IC50 value of 1 X 10−8 and 1 X 10−5 mol/L in the outer and inner layers, respectively.

Characterization of neurotensin binding sites on rat mesencephalic cells in primary culture

Many studies have reported the presence of high amounts of neurotensin (NT) binding sites in the mesencephalon of adult rat, and their possible role in mediating the effects of the peptide on the activity of mesencephalic dopaminergic neurons. In the present study, we demonstrate the presence of NT sites in primary cultures of embryonic rat mesencephalic cells. On these cells, a single class of high affinity 125I-NT binding sites was observed. The value of the apparent affinity constant (0.3 nM) did not show any significant change throughout time, from 3 to 14 days in culture. The number of sites, however, increased until day 11 and decreased thereafter. Acetylneurotensin (8-13), NT and neuromedin N were potent competitors of 125I-NT binding, while NT (1-10), NT (1-11) and levocabastine were uneffective. These results indicate that the sites detected in the mesencephalic cultures share common binding properties with the high-affinity NT sites already described in adult rat brain. The neuronal localization of the NT sites was suggested by their presence in neuron-enriched serum-free cultures and their absence in glial cultures. Autoradiographic studies confirmed the cellular localization of NT sites and indicated that, under our experimental conditions, cells labeled by 125I-NT represented 0.14% of the initially plated cell number. Taken together, these results show that the development of mesencephalic neurons in primary culture is associated with an increased expression of NT binding sites. Since such cultures have been shown previously to contain functional dopaminergic neurons, we suggest that they could provide a good model to investigate the modulation of the activity of these neurons by NT.

Potent cocaine analogs inhibit [3H]dopamine uptake in rat mesencephalic cells in primary cultures: pharmacological selectivity of embryonic cocaine sites

The cellular localization of the cocaine binding sites in primary cultures of embryonic rat mesencephalic cells was previously reported to differ from that observed in adult rat brain. In order to know whether this different localization was associated with a different pharmacological selectivity, we tested the effect of new cocaine analogs on tritiated dopamine ([3H]DA) uptake in primary cultures of rat embryonic mesencephalic cells. In these cultures, [3H]DA was taken up by a nomifensine-sensitive, but desipramine and fluoxetine-insensitive process, reflecting selective uptake by the dopaminergic transporter. 3 beta-(4-Chlorophenyl)tropan-2 beta-carboxylic acid methyl ester (RTI-COC-31) was by far the most potent inhibitor of the [3H]DA uptake, presenting an IC50 of 3.8 nM, while the corresponding analog with an unsubstituted phenyl ring (WIN 35,065-2) was 38 times less potent. The enantiomer of WIN 35,065-2, namely WIN 35,065-3, was 30 times less potent than the former. A similar pattern was found for the relative ability of these compounds to inhibit binding of the radiolabeled cocaine derivative [125I]RTI-55 to membranes prepared from mesencephalic cultures. The order of potencies found for the three cocaine analogs on mesencephalic cultures was similar to that previously obtained in [3H] WIN 35,428 binding experiments and [3H]DA uptake inhibition in adult rat striatum, suggesting that the pharmacological selectivity of cocaine sites functionally related to the DA transporter in cultured embryonic neurons does not differ from that obtained in adult rat brain.

Hyperprolactinemia-induced modifications in vasoactive intestinal peptide binding site densities in the rat central nervous system and pituitary gland: evidence for an interaction between estradiol-17β and prolactin effects

Hyperprolactinemia was induced in ovariectomized rats by implanting estradiol-17 beta pellets, grafting extrapituitaries, or by a combination of both treatments. Subsequently, the effect of increasing plasma prolactin levels on both central and pituitary receptors for vasoactive intestinal peptide (VIP), a neuropeptide known to stimulate prolactin release was investigated. The results obtained by quantitative autoradiography show that the density of VIP binding sites is modified in restricted areas of the central nervous system (striatum, several cortical, thalamic and limbic structures) and in the pituitary in hyperprolactinemic animals. The present results suggest that changes in plasma prolactin levels may control VIP receptor site density in both brain and pituitary. Moreover, direct effects of estradiol-17 beta and possible interactions between estradiol-17 beta and prolactin are observed on both brain and pituitary VIP binding sites.

Autoradiographic Localization of Brain Peptide Receptors at the Electron Microscopic Level

Neurotransmitters and related drugs exert their biochemical, electrophysiological, and, ultimately, behavioral effects by acting upon specific receptor molecules embedded in neuronal and, in some cases, perhaps also glial plasma membranes. This interaction involves: (1) recognition of a specific binding site on the surface of the receptor and (2) translation of the recognition information into a response signal. Pioneering studies by Clark (1933) demonstrated that the binding of drugs (ligands) to receptors was reversible, obeyed the law of mass action, and occurred at very low concentrations of ligand. Considerable progress in our understanding of receptor mechanisms was later to emerge from the development of sensitive methods for measuring the receptor-specific binding of radiolabeled ligand probes (Snyder and Bennett, 1976). Not only did the availability of such methods greatly facilitate the biophysical, pharmacological, and molecular characterization of receptors for neurotransmitters, but it also made possible their localization in situ, using autoradiographic techniques.