Localization and characterization of binding sites for vasopressin and oxytocin in the brain of the guinea pig.

Using autoradiography on film, specific binding sites for arginine-vasopressin (AVP) and for oxytocin (OT) were localized in various areas of the brain of adult male guinea pigs. Vasopressin binding sites were detected with [3H]AVP or with [125I]VPA, a recently synthetized linear vasopressin antagonist radiolabeled with 125I. [125I]VPA and [3H]AVP yielded similar results, thus suggesting that AVP binding sites present in the guinea pig brain are V1 type receptors. Supporting evidence on this was obtained in competing studies using structural analogues allowing to discriminate V1 receptors from V2 and from OT receptors. Oxytocin binding sites were labeled with [3H]OT or with the iodinated OT antagonist [125I]OTA; both ligands yielded similar results. The localization in the guinea pig brain of AVP binding sites differed from that of OT binding sites. AVP binding sites were mainly detected in the olfactory bulb and throughout the cerebral cortex. Oxytocin binding sites were most noticeable in the hypothalamic ventromedial nucleus, in the amygdaloid complex and in restricted areas of the cerebral cortex. A comparison of the present data with those previously described in the rat, the mouse, the human and the hamster brain suggests that similar binding sites are present in these species, but that their anatomical distribution differs markedly. These data are discussed in relation to immunocytochemical and electrophysiological data which suggest that binding sites detected by autoradiography may represent, at least in part, functional neuronal receptors.     

Ontogeny of oxytocin receptors in rat forebrain: a quantitative study

The ontogeny of oxytocin receptors in rat forebrain was studied using the selective oxytocin receptor antagonist 125I-d(CH2)5[Tyr(Me)2, Thr4, Tyr-NH29]OVT [( 125I]-OTA). With in vitro receptor autoradiography, binding wa noted on the first postnatal day in dorsal subiculum and thalamus. On postnatal days 5-18, intense labeling was evident in posterior cingulate cortex, dorsal subiculum, lateral septum, and the CA1 subfield of hippocampus. Of these regions only the lateral septum expressed oxytocin receptors in adult brain. Competition studies on coronal sections through posterior cingulate, septum, and dorsal subiculum at P10 demonstrated that transient binding sites in these areas were indeed oxytocin selective (OXY greater than AVP greater tha V1 greater than V2). Result of saturation studies on cingulate membranes from 10-day-old pups agreed favorably with previous reports of the kinetics of [125I]-OTA binding to adult oxytocin receptors (Kd = 0.1 nM in P10 cingulate cortex vs. 0.07 nM for adult ventral subiculum). In contrast to these evanescent developmental sites, oxytocin receptors in the bed nucleus of the stria terminalis and the ventromedial nucleus of the hypothalamus only appeared in adulthood, presumably in response to the surge of gonadal steroids at puberty.     

Castration reduces vasopressin receptor binding in the hamster hypothalamus

A recently developed ligand with very high affinity and selectivity for the vasopressin (AVP) V_1a receptor subtype (i.e. [¹²⁵I]Linear AVP antagonist ([¹²⁵I]-LinAntag) was used to describe the distribution of AVP binding sites in the hamster brain, and to determine whether AVP receptor binding was influenced by testicular hormones in sites involved in the regulation of steroid-dependent social behaviors. These studies demonstrated [¹²⁵I]LinAntag binding in regions of the hamster brain which have not been previously identified with other AVP ligands. In addition, testicular hormones were found to alter [¹²⁵I]LinAntag binding in two distinct regions, the posterior lateral preoptic-anterior lateral hypothalamic continuum and the posterior ventrolateral hypothalamic nucleus and adjacent tuberal area.     

Localization and pharmacological characterization of high affinity binding sites for vasopressin and oxytocin in the rat brain by light microscopic autoradiography

Sites which bind tritiated vasopressin (AVP) with high affinity were detected in the brain of male, adult rats, by light microscopic autoradiography. Their anatomical localization differed markedly from that of high affinity binding sites for tritiated oxytocin (OT) determined in the same animal. Co-labelling was minimized by using low concentrations of [3H]AVP and [3H]OT. Binding of the former occurred predominantly in several structures of the limbic system (septum, amygdala, bed nucleus of the stria terminalis, accumbens nucleus), in two hypothalamic nuclei (suprachiasmatic and dorsal tuber) and in the area of the nucleus of the solitary tract. Binding of OT was evidenced in the olfactory tubercle, the ventromedial hypothalamic nucleus, the central amygdaloid nucleus and the ventral hippocampus. The ligand specificity of the binding sites was assessed in competition experiments. Synthetic structural analogues were used, allowing to discriminate OT receptors (OH[Thr4,Gly7]OT) from V2 receptors (dDAVP and d[Tyr(Me)2]VDAVP), V1 receptors ([Phe2,Orn8]VT) and V1b receptors (desGly9d(CH2)5AVP). Our main conclusions are, firstly, that AVP and OT binding sites can be readily distinguished, and that there is virtually no overlap in their distribution in the rat brain. Second, we showed that the sites which bind AVP with high affinity in the brain are V1 receptors, different both from the renal V2 receptors and from the anterior pituitary V1b receptors. Our results support the conjecture that AVP and OT play a role in interneuronal communication in the brain.     

Distribution of an alpha-bungarotoxin-binding cholinergic nicotinic receptor in rat brain

Cholinergic nicotinic receptors in rat brain were demonstrated by the use of the potent nicotinic antagonist [125I]alpha-bungarotoxin [125I]alpha-Btx). Biochemical studies on binding of [125I]alpha-Btx to rat hippocampal homogenates revealed saturable binding sites which are protected by nicotine, D-tuborcurarine and acetylcholine but not by atropine or oxotremorine. The hippocampus and hypothalamus displayed relatively high [125I]alpha-Btx specific binding whereas the cerebellum was devoid of specific binding. Other regions displayed intermediate binding levels. Analysis of the regional distribution of [125I]alpha-Btx binding by autoradiography of frontal brain sections revealed high labeling in the hippocampus, hypothalamic supraoptic, suprachiasmatic and periventricular nuclei, ventral lateral geniculate and the mesencephalic dorsal tegmental nucleus. It is suggested that the limbic forebrain and midbrain structures as well as sensory nuclei are the main nicotinic cholinoceptive structures in the brain.     

6β-[125Iodo]-3, 14-dihydroxy-17-methyl-4, 5α-epoxymorphinan ([125I]IOXY-AGO): A potent and selective radioligand for opioid μ receptors

The recent cloning and expression of an opioid μ receptor has opened up new opportunities for research in opioid pharmacology. The relatively low level of transient receptor expression in COS cells emphasizes the need for radioligands with high specific activity and low nonspecific binding with which to label receptors. In addition, recent data indicating that agonists and antagonists bind to different domains on the same receptor protein indicate the utility of having both agonist and antagonist radioligands available for the study of opioid receptor mechanisms. Previous studies characterized the binding of the. opioid antagonist 6β-[¹²⁵iodo]-3,14-dihydroxy-17-cyclopropylmethyl-4,5α-epoxymorphinan ([¹²⁵I]IOXY) and showed that this naltrexone analog labels μ and K₂ receptors in rat and guinea pig brain with high affinity and low nonspecific binding. In the present study, we synthesized the agonist congener of IOXY, 6β-iodo-3,14-dihydroxy-17-methyl-4,5α-epoxymorphinan. We named this novel agent IOXY-AGO for IOXY-agonist. Competition binding studies showed that IOXY-AGO has high affinity for δ receptors (Ki = 0.28 nM) and lower affinity for δ (Ki = 18.7 nM) and K₁ (Ki = 33.9 nM), K_2a (Ki = 38.4 nM) and K_2b (Ki = 58.2 nM) binding sites. IOXY-AGO was radioiodinated to a specific activity of 2,200 Ci/mmol. [¹²⁵I]IOXY-AGO binding was rapid, readily reversible, and characterized by low nonspecific binding. Equilibrium binding studies showed that it labeled a single class of binding sites (Kd = 1.46 nM, Bmax = 112 fmol/mg protein) with the characteristics of an opioid μ receptor. Receptor autoradiography experiments showed that [¹²⁵I]IOXY-AGO labeled binding sites with the anatomical distribution of μ receptors. Viewed collectively, these studies suggest that [¹²⁵I]IOXY-AGO will be a useful radioligand for characterizing opioid μ receptors. © 1995 Wiley-Liss, Inc.     

Autoradiographic visualization and characteristics of [125I]bradykinin binding sites in guinea pig brain.

The present study was undertaken to localize and characterize bradykinin (BK) binding sites in 10 microns serial sections of guinea pig brain by in vitro quantitative receptor autoradiography. Specific binding of [125I-Tyr8]bradykinin ([125I]BK) was localized in the medulla oblongata to the regions of the nucleus of the solitary tract (nTS), the area postrema (AP), the dorsal motor nucleus of the vagus (X) and the caudal subnucleus of the spinal trigeminal nucleus. No significant specific [125I]BK binding was seen in other brain regions. The specific binding (85-90% of total binding) was of high affinity and saturable with a KD of 73.5 +/- 9.9 pM and a Bmax of 27.8 +/- 1.9 amol per mm2 of tissue. In competition studies, the rank order of potencies was: BK greater than Met-Lys-BK greater than Lys-BK much greater than Des-Arg9-BK. The B2 receptor antagonist D-Arg0-Hyp3-Thi5,8-D-Phe7-BK inhibited [125I]BK binding with a Ki value of 3.5 +/- 1.5 nM while Des-Arg9-[Leu8]-BK, a B1 receptor antagonist did not significantly inhibit [125I]BK binding in concentrations up to 10 microM. Our finding of specific high affinity [125I]BK binding sites in the nTS, AP and the X is important because these brain areas are known to be involved in central cardiovascular regulation. Moreover, our results suggest that the specific [125I]BK binding sites in the guinea pig medulla are of the bradykinin B2 receptor type.     

Evidence for heterogeneity of muscarinic receptors in the mollusc Pleurobranchaea

The properties of the specific binding of the muscarinic antagonist [125I]3-quinuclidinyl-4-iodobenzilate ([125I]4IQNB] to nervous tissue of Pleurobranchaea california were characterized. The specific binding of [125I]4IQNB to Pleurobranchaea nervous tissue was characterized by its high affinity (Kd = 0.61 +/- 0.11 nM) and saturability (Bmax = 602 +/- 46 fmol/mg protein). A comparison of the numbers of binding sites recognized by [125I]4IQNB and l-[3H]QNB in nervous tissue of three invertebrate species indicated that in Aplysia and Cancer magister (crab) ganglia membranes the two radioligands labeled comparable numbers of binding sites; however, in Pleurobranchaea membranes l-[3H]QNB recognized only a subpopulation (8-10%) of the total number of [125I]4IQNB binding sites. The disparity in the numbers of binding sites labeled by these radioligands was consistent with our finding of a heterogeneity of muscarinic antagonist binding sites in l-QNB competition experiments in Pleurobranchaea. Computer-assisted analysis of l-QNB competition of [125I]4IQNB specific binding demonstrated that these data were best described by a two-site model with high- and low-affinity sites for l-QNB. The high-affinity site recognized by l-QNB possessed an IC50 value of 0.2 nM and comprised 18% of the total specific binding, while the lower affinity site had an IC50 value of 55.6 nM and comprised the remaining 82% of the total population of [125I]4IQNB recognition sites. The IC50 value for l-QNB at the high-affinity site in Pleurobranchaea membranes is in excellent agreement with Kd values for l-[3H]QNB labeling of classical muscarinic receptors in a variety of invertebrate and vertebrate species.     

Pharmacological characterization of oxytocin-binding sites in rat spinal cord membranes: comparison with embryonic cultured spinal cord neurones and astrocytes

Detection and pharmacological characterization of OT-binding sites were performed on 12-day-old rat spinal cord membranes and on embryonic cultured spinal neurones and astrocytes after 12 days in culture. In neurone-enriched cultures, OT-binding sites were detected by autoradiography on cells morphologically comparable to neurone-specific enolase immunoreactive cells. In astrocyte cultures, as shown by combination of autoradiography and immunocytochemistry, OT-binding sites were detected on cells expressing the glial fibrillary acidic protein (a specific astrocytic marker). The pharmacological characterization was assessed by binding studies performed with a highly specific radioiodinated OT antagonist on postnatal rat spinal cord membranes and on embryonic cultured spinal cord neurones and astrocytes. The saturation studies suggested the presence of a single class of binding sites of high affinity for the OT antagonist on spinal membranes and cultured cells, as already described in the rat for central and peripheric OT receptors. The competition studies indicated that on spinal membranes, OT and AVP had the same high affinity, as classically described, whereas on cultured cells, AVP had a lower affinity, suggesting that culture conditions may influence the pharmacology of the spinal OT-binding sites. Involvement of NEM- and Gpp[NH]p-insensitive G-proteins in the coupling of the spinal OT-binding sites with the effector system was evidenced on 12-day-old rat spinal membrane preparations and on neurone and astrocyte cultures.     

An autoradiographic study of the distribution of binding sites for the novel alpha7-selective nicotinic radioligand [3H]-methyllycaconitine in the mouse brain.

[3H]-Methyllycaconitine ([3H]-MLA) is a new radioligand with selectivity for alpha7-type neuronal nicotinic acetylcholine receptors (nAChRs). In our previous study [Davies, A.R.L., Hardick, D.J., Blagbrough, I.S., Potter, B.V.L., Wolstenholme, A.J. & Wonnacott, S. (1999) Neuropharmacology, 38, 679-690], this radioligand labelled a single class of site in rat brain membranes; its pharmacology and distribution in crudely dissected brain regions closely paralleled that of the well-established alpha7-ligand [125I]-alpha-bungarotoxin. However, a small population of [3H]-MLA binding sites was apparently insensitive to alpha-bungarotoxin. Here we have extended the study to mouse brain, using autoradiography to examine the distribution of [3H]-MLA and [125I]-alpha-bungarotoxin binding sites. [3H]-MLA labelled a single class of site in mouse brain membranes with a KD of 2.2 nM and a Bmax of 45.6 fmol/mg protein. Specific binding, defined by unlabelled MLA (Ki = 0.69 nM), was completely inhibited by (-)-nicotine (Ki = 1.62 microM), whereas alpha-bungarotoxin inhibited only 85% of specific binding (Ki = 3.5 nM). The distributions of [125I]-alpha-bungarotoxin and [3H]-MLA binding sites were compared by autoradiography, and binding was quantitated in 72 brain regions. Binding of both radioligands was highly correlated, with highest densities in the dorsal tegmental nucleus of the pons, colliculi and hippocampus. Serial sections labelled with [3H]-MLA in the absence or presence of unlabelled MLA or alpha-bungarotoxin provided no evidence for any alpha-bungarotoxin-resistant binding. The results are discussed in terms of binding sites that are inaccessible to alpha-bungarotoxin in membrane preparations. This study demonstrates the utility of [3H]-MLA for characterization of alpha7-type nicotinic receptors in mammalian brain, and suggests that it labels a population identical to that defined by [125I]-alpha-bungarotoxin.     

Localization of high-affinity binding sites for oxytocin and vasopressin in the human brain. An autoradiographic study

Sites which bind oxytocin and vasopressin with high affinity were detected in the brain and upper spinal cord of 12 human subjects, using in vitro light microscopic autoradiography. Tissue sections were incubated with tritiated vasopressin, tritiated oxytocin or an iodinated oxytocin antagonist. The ligand specificity of binding was assessed with unlabelled vasopressin or oxytocin in excess, as well as in competition experiments using synthetic structural analogues. The distribution of vasopressin binding sites differed markedly from that of oxytocin binding sites in the forebrain, while there was overlap in the brainstem. Vasopressin binding sites were detected in the dorsal part of the lateral septal nucleus, in midline nuclei and adjacent intralaminar nuclei of the thalamus, in the hilus of the dentate gyrus, the dorsolateral part of the basal amygdaloid nucleus and the brainstem. The distribution of oxytocin binding sites in the brainstem has been recently reported (Loup et al., 1989). Oxytocin binding sites were also observed in the basal nucleus of Meynert, the nucleus of the vertical limb of the diagonal band of Broca, the ventral part of the lateral septal nucleus, the preoptic/anterior hypothalamic area, the posterior hypothalamic area, and variably in the globus pallidus and ventral pallidum. The presence of oxytocin and vasopressin binding sites in limbic and autonomic areas suggests a neurotransmitter or neuromodulator role for these peptides in the human central nervous system. They may also affect cholinergic transmission in the basal forebrain and consequently play a role in Alzheimer's disease.     

125I]Iodomethyllycaconitine binds to alpha7 nicotinic acetylcholine receptors in monkey brain

We examined the binding of the novel nicotinic acetylcholine receptor (nAChR) ligand [125I]iodomethyllycaconitine (iodoMLA) in the brains of M. cynomologous (macaque) monkeys. [125I]iodoMLA bound throughout the brain with the greatest density in the thalamus and moderate intensity in the basal ganglia and cortical regions. The Kd and Bmax in whole brain tissue were similar whether 1 mM nicotine (Kd 33.25 +/- 15.17 nM, Bmax 5.80 +/- 1.06 fmol/mg) or 2 microM of the alpha7-selective antagonist alpha-bungarotoxin (Kd 46.12 +/- 18.45 nM, Bmax 6.30 +/- 1.06 fmol/mg) was used for nonspecific binding. The subtype-selectivity of this ligand was further studied with competition binding studies using nicotine, alpha-bungarotoxin and noniodinated MLA. Each ligand completely inhibited [125I]iodoMLA binding throughout the monkey brain, with Ki values of 2.23 +/- 0.85 microM for nicotine, 2.72 +/- 1.71 nM for alpha-bungarotoxin and 1.83 +/- 0.35 nM MLA in the caudate and 2.03 +/- 1.14 microM, 2.65 +/- 0.86 nM and 3.32 +/- 0.71 nM, respectively, in the putamen. The alpha3beta2/alpha6*-selective antagonist alpha-conotoxin MII failed to inhibit [125I]iodoMLA binding in any brain region. In monkeys with cognitive deficits resulting from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration, [125I]iodoMLA binding was significantly increased in the striatum, similar to results previously observed for [125I]alpha-bungarotoxin. These results suggest that, under the present experimental conditions, [125I]iodoMLA was selective for alpha7-containing nAChRs and did not bind to alpha6-containing nAChRs. This radioligand may be a useful tool for selectively imaging alpha7-containing nAChRs in vivo.     

An 125I-labeled binding probe for the muscarinic cholinergic receptor.

We describe the synthesis and characterization of [125I]3-quinuclidinyl-(3-iodo-4-hydroxy-benzilate), a binding probe for the muscarinic cholinergic receptor of high specific radioactivity. The binding isotherm of this 125I-labeled compound to a rat synaptic plasma membrane-enriched fraction consists of two components: a 'specific' component which is saturable and closely fits hyperbolic binding to a single class of sites as evaluated by Scatchard analysis (Kd = 1.5 to 3 nM), and a linear component which may be measured directly by preincubating membranes in 0.1 microM 3-quinuclidinyl-benzilate, the most potent muscarinic antagonist known. The specific binding of [3-3H]3-quinuclidinyl-benzilate and [125I]3-quinuclidinyl-(3-iodo-4-hydroxy-benzilate) to rat brain subcellular fractions is parallel in myelin, synaptic plasma membrane and mitochondrial fractions with a 3--4-fold enrichment observed in synaptic plasma membrane over crude mitochondrial fractions. The concentrations of muscarinic antagonists required to block [125I]3-quinuclidinyl-(3-iodo-4-hydroxy-benzilate) binding parallel that reported for tritiated binding probes and are consistent with physiological measurements of their dissociation constants. Because of the high specific radioactivity of [125I]3-quinuclidinyl-(3-iodo-4-hydroxy-benzilate), this iodinated binding probe should prove useful in assaying low levels of muscarinic receptor in tissue culture and other biological sources.     

Distribution of an α-bungarotoxin-binding cholinergic nicotinic receptor in rat brain

Cholinergic nicotinic receptors in rat brain were demonstrated by the use of the potent nicotinic antagonist [¹²⁵I]α-bungarotoxin ([¹²⁵I]α-Btx). Biochemical studies on binding of [¹²⁵I]α-Btx to rat hippocampal homogenates revealed saturable binding sites which are protected by nicotine, d-tubocurarine and acetylcholine but not by atropine or oxotremorine. The hippocampus and hypothalamus displayed relatively high [¹²⁵I]α-Btx specific binding whereas the cerebellum was devoid of specific binding. Other regions displayed intermediate binding levels. Analysis of the regional distribution of [¹²⁵I]α-Btx binding by autoradiography of frontal brain sections revealed high labeling in the hippocampus, hypothalamic supraoptic, suprachiasmatic and periventricular nuclei, ventral lateral geniculate and the mesencephalic dorsal tegmental nucleus. It is suggested that the limbic forebrain and midbrain structures as well as sensory nuclei are the main nicotinic cholinoceptive structures in the brain.     

Oxytocin Receptor Binding in Female Prairie Voles: Endogenous and Exogenous Oestradiol Stimulation

Previous studies have demonstrated that oxytocin receptors in specific nuclei of rat forebrain are regulated by gonadal steroids. The current study used in vitro receptor autoradiography to investigate the distribution and regulation of oxytocin receptors in the forebrain of the female prairie vole (Microtus ochrogaster). In contrast to rats, in female prairie voles gonadal steroid secretion and oestrus behaviour result from male chemosignal stimulation and ovulation is induced by mating. Thus, the prairie vole brain provides an opportunity for investigating links between environmental stimuli, gonadal steroids and oxytocin receptors. Using a selective oxytocin receptor ligand [¹²⁵l]d(CH²)⁵[Tyr(Me)²,Tyr-NH₂⁹]ornithine vasotocin ([¹²⁵I]OTA), specific binding was found in several regions including the anterior olfactory nucleus, the ventromedial nucleus of the hypothalamus, the bed nucleus of the stria terminalis, the amygdala and several cortical areas. Following ovariectomy, oestradiol benzoate (10 μg) administration increased oxytocin receptor binding 100% in the anterior olfactory nucleus, but did not affect receptors in other regions. Gonadallyintact females, exposed to male chemosignals, had significant increases in both endogenous oestradiol levels and anterior olfactory nucleus oxytocin receptor binding relative to gonadally-intact females unexposed to male chemosignals. Following prolonged exposure to males with ad libitum mating, [¹²⁵I]OTA receptor binding decreased to the levels found in unstimulated females. These results demonstrate that increases in oestrogen levels, of either exogenous or endogenous origin, can modulate oxytocin receptors in the brains of female prairie voles. In contrast to rats, oestrogen in female prairie voles appears to affect receptors in the anterior olfactory nucleus rather than the hypothalamus. We suggest that the species differences in oxytocin receptor distribution and gonadal steroid responsiveness reflect variations in reproductive physiology and possibly behaviour.     

Characterization of vasopressin receptor in rat lung

This study characterized rat lung membrane arginine vasopressin (AVP) receptors in detail. Specific binding of [3H]AVP to rat lung membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with a Kd of 0.45 nM and a Bmax of 76.6 fmol/mg protein. Competitive inhibition of [3H]AVP binding showed that neurohypophysial hormones as well as their synthetic analogues displaced [3H]AVP in a concentration-dependent manner. The order of potencies for the native peptides was: AVP > lysine vasopressin = arginine vasotocin > oxytocin. Furthermore, potent V1A receptor antagonists, d(CH2)5Tyr(Me)AVP and dPTyr(Me)AVP, showed high affinity for lung membranes. In contrast, the V2 receptor agonist, dDAVP, and the specific oxytocin receptor agonist, [Thr4,Gly7]oxytocin, did not affect AVP binding. These results suggest that the lung contains the V1A receptor subtype. The lung membrane AVP receptor characterized in this study may play an important role in mediating the physiological effects of AVP in the lung.     

VIP receptor subtypes in mouse cerebral cortex: evidence for a differential localization in astrocytes, microvessels and synaptosomal membranes.

The binding characteristics of a monoiodinated form of vasoactive intestinal peptide (M-[125I]VIP) to the membranes of astrocytes, intraparenchymal microvessels and synaptosomes were analyzed in mouse cerebral cortex. Binding to astrocytes, studied in primary cultures, indicates the presence of a single class of high affinity binding sites with a Kd of 3.3 nM and a Bmax of 565 fmol/mg protein. The structurally related peptide secretin does not compete for sites labeled by M-[125I]VIP. In cultured astrocytes, VIP has been previously shown to promote glycogenolysis. Secretin, despite its lack of interaction with sites labeled by M-[125I]VIP, stimulates glycogenolysis with an EC50 of 0.5 nM, thus demonstrating the presence in astrocytes of functional secretin receptors independent from those for VIP. Trypsinization of the primary astrocyte cultures followed by replating as secondary cultures, reveals a second class of low affinity binding sites, with a Kd of 41.3 nM and a Bmax of 881 fmol/mg protein. Secretin does not compete for this class of low affinity binding sites either. Binding of M-[125I]VIP to intraparenchymal microvessels reveals the presence of two classes of binding sites with Kd of 1.4 and 30.3 nM, and Bmax of 7.1 and 73.8 pmol/mg protein, respectively. Similar to what is observed in primary or secondary astrocyte cultures, secretin does not interact with these sites. In this cell type VIP stimulates cAMP formation with an EC50 of 18 nM, while secretin is ineffective. Finally, in agreement with previous reports in rat and guinea pig cerebral cortex, two classes of binding sites are observed in synaptosomal membranes: a high affinity class with a Kd of 4.9 nM and a Bmax of 316 fmol/mg protein, and a low affinity class with a Kd of 42.8 nM and a Bmax of 1578 fmol/mg protein. In contrast to what is observed in non-neuronal membranes, in synaptosomal membranes, secretin effectively competes for sites labeled by M-[125I]VIP with an EC50 of approximately 150 nM. These results indicate that secretin may represent a useful tool to discriminate between neuronal and non-neuronal VIP binding sites, since it competes with M-[125I]VIP exclusively for the neuronal class of binding sites.     

Neurophysins, rather than Receptors, are Involved in [3H]Oxytocin and [3H]Vasopressin Binding Detected by Autoradiography in the Hypothalamo-Neurohypophyseal System

The goal of the present experiments was to analyse the binding of oxytocin (OT) and vasopressin (VP) in the hypothalamo-neurohypophyseal system to determine whether [³H]OT and [³H]VP binding in this system involved interaction with receptor sites or with neurophysins. Using quantitative autoradiography, several experiments were performed to compare [³H]OT- and [³H]VP-binding characteristics in this system and in brain areas containing identified receptor sites. Saturation experiments indicated much lower affinity of [³H]OT and [³H]VP binding in the magnocellular nuclei and neural lobe than on brain receptors. Competition experiments using selective ligands indicated interaction with neurophysins rather than with receptors in the hypothalamo-neurohypophyseal system. This system was never labelled in the presence of a [¹²⁵I]OT antagonist, a selective OT receptor ligand. In contrast with receptors elsewhere in the brain, the magnocellular nuclei were labelled by [³H]OT and [³H]VP in the absence of MgCI₂. In the pituitary neural lobe, density of binding sites was moreover obviously related to the amount of neurosecretory granules, as seen in acutely dehydrated rats. Taken together, these data strongly suggest that in the hypothalamo-neurohypophyseal system [³H]OT and [³H]VP bind to neurophysins rather than to specific receptors.     

Binding sites for pituitary adenylate cyclase activating polypeptide (PACAP): comparison with vasoactive intestinal polypeptide (VIP) binding site localization in rat brain sections.

Pituitary adenylate cyclase activating polypeptide (PACAP) is structurally similar to vasoactive intestinal polypeptide (VIP). We investigated the characteristics and topographical distribution of [125I]PACAP binding sites compared with those of [125I]VIP binding sites in the rat brain. Radiolabeled PACAP and VIP showed highly specific binding to sections at the level of the dorsal hippocampus. The specific binding of [125I]PACAP was 10 times higher than that of [125I]VIP in hippocampal sections. [125I]PACAP binding was scarcely displaced by unlabeled VIP, while [125I]VIP binding was effectively displaced by unlabeled PACAP. Therefore, PACAP binding sites may reflect both PACAP specific binding sites and VIP/PACAP binding sites. However, the amount of VIP/PACAP binding sites was negligibly low. Autoradiography revealed that [125I]PACAP binding sites were dense in the piriform cortex, diagonal band, accumbens nucleus, anterior part of the striatum, hippocampal formation, habenular nucleus, lateral hypothalamic area, superior colliculus and dorsal raphe nucleus. Moderate to high labeling was observed in the medial septal nucleus, olfactory tubercle, caudal part of the striatum, most parts of the thalamus, supraoptic and periventricular hypothalamic nuclei, central gray, substantia nigra pars compacta, locus coeruleus, pontine reticular nucleus and cerebellum. Distribution pattern was remarkably different from that of [125I]VIP binding sites in the hippocampal formation, lateral hypothalamic area, substantia nigra pars compacta, pontine reticular nucleus and cerebellum. The present results suggest that PACAP may have a physiological role in the regulation of the central nervous system.     

Binding affinities of oxytocin,vasopressin and Manning compound at oxytocin and V1a receptors in male Syrian hamster brains

Oxytocin (OT) and arginine vasopressin (AVP), as well as synthetic ligands targeting their receptors (OTR, V1aR), are used in a wide variety of research contexts, although their pharmacological properties are determined in only a few species. Syrian hamsters (Mesocricetus auratus) have a long history of use as a behavioural and biomedical model for the study of OT and AVP and, more recently, hamsters have been used to investigate behavioural consequences of OT‐mediated activation of V1aR. We aimed to determine the binding affinities of OT, AVP and the selective V1aR antagonist, Manning compound, for OTR and V1aR in hamster brains. We performed saturation binding assays to determine the Kd values for the selective OTR and V1aR radioligands, [¹²⁵I]ornithine vasotocin analogue and [¹²⁵I]linear vasopressin antagonist. We then performed competition binding assays to determine K_i values for OT, AVP and Manning compound at both the OTR and V1aR. We found that OT and AVP each had the highest affinity for their canonical receptors (OT‐OTR Ki = 4.28 [95% confidence interval (CI) = 2.9‐6.3] nmol L^?1; AVP‐V1ar Ki = 4.70 [95% CI = 1.5‐14.1] nmol L^?1) and had the lowest affinity for their non‐canonical ligands (OT‐V1aR = 495.2 [95% CI = 198.5‐1276] nmol L^?1; AVP‐OTR Ki = 36.1 [95% CI = 12.4‐97.0] nmol L^?1). Manning compound had the highest affinity for the V1aR (MC‐V1aR Ki = 6.87 [95% CI = 4.0‐11.9] nmol L^?1; MC‐OTR Ki = 213.8 [95% CI = 117.3‐392.7] nmol L^?1), although Manning compound was not as selective for the V1aR in hamsters as has been reported for the receptor in rats. When comparing these data with previously published work, we found that the promiscuity of the V1aR in hamsters with respect to OT and AVP binding is more similar to the promiscuity of the human V1aR than to the rat V1aR receptor. Moreover, the selectivity of OT at hamster receptors is more similar to the selectivity of OT at human receptors than the selectivity of OT at rat receptors. These data highlight the importance of determining the pharmacological properties of behaviourally relevant compounds in diverse model species.