A reduction in the concentration of immunoreactive corticotropin, malanotropin and lipotropin in the brain of the aging rat

We have previously shown that the hypothalamic concentration of immunoreactive α-melanotropin (α-MSH_i) is markedly lower in the aging female rat than in the young rat. The current view is that α-MSH is derived from corticotropin (ACTH), and ACTH, in turn, is derived from a large molecular-weight precursor (pro-opiocortin); pro-opiocortin also serves as the precursor to β- and γ-lipotropin (LPH)^{10, 12, 22}. To ascertain if the age-related reduction in the concentration of α-MSH_i may be a result of a decline in the production of pro-opiocortin, we determined the content of immunoreactive ACTH (ACTH_i), α-MSH (α-MSH_i), γ-LPH (γ-LPH_i), and protein, in 3 regions of the brain of young (4 months) and old (26–28 months) female rats: the medial basal hypothalamus (MBH, the region containing the perikarya of the ACTH/MSH/LPH neurons^{9, 23, 28, 36}), the preoptic anterior hypothalamus (POA), and the thalamus (regions containing axons of these neurons). The concentration of ACTH_i, α-MSH_i (mol/mg protein), or γ-LPH_i (U/mg protein) in the MBH of old rats was 30–50% of that in the MBH of young rats. Moreover, the concentration of ACTH_i, α-MSH_i or γ-LPH_i in the POA and thalamus of old rats was also lower than that in the POA and thalamus of young rats. Based on these findings, we propose that aging causes a reduction in the production of pro-opiocortin in the brain of the female rat and that such a change may be related to the altered function of the brain of the aged.     

Peptides and the conversion of [H]tyrosine to catecholamines: Effects of ACTH-analogs, melanocyte-stimulating hormones and lysine-vasopressin

ACTH_4–10, [D-Phe₇]ACTH_4–10, ACTH_1–24, α-MSH and lysine-vasopressin (LVP) were tested for their effects on the cerebral content and metabolism of dopamine (DA) and norepinephrine (NE). CD-1 mice were injected daily for 3 days with equimolar concentrations of long-acting preparations of the peptides. Twenty-four hours after the third peptide treatment, mice were injected with [³H]tyrosine and the specific activities of tyrosine, DA and NE were measured 10 min later.ACTH_1–24, ACTH_4–10 or [D-Phe₇]ACTH_4–10 stimulated the conversion of [³H]tyrosine to DA but did not alter the cerebral dopamine content, suggesting that the turnover of dopamine was increased. α-MSH, β-MSH or LVP did not show this effect. Since ACTH_4–10 has low steroidogenic activity it appears that the effect on [³H]DA metabolism was a direct effect of the peptide and not mediated by increased corticosterone secretion.None of the peptides had any detectable effect on the metabolism of [³H]NE. ACTH_1–24 but no other peptide decreased the cerebral concentration of NE. In mice adrenalectomized one day before the first peptide injection, ACTH_4–10 did not alter the specific activity of DA or NE.     

Subcellular localization of immunoreactive α-melanocyte stimulating hormone in human brain

The regional and subcellular distribution of immunoreactive α-melanocyte stimulating hormone (α-MSH₁) in the post mortem adult human brain was investigated. α-MSH_i was highly concentrated in medial basal hypothalamic tissue (1.02 ng/mg protein). Lower levels of α-MSH_i were present in the optic chiasm and mammillary bodies, 0.08 and 0.11 ng/mg protein, respectively. The concentrations of α-MSH_i in cerebellum and frontal cerebral cortex were 1/1,000th that of the medial basal hypothalamus. When medial basal hypothalamic homogenates were subjected to discontinuous or continuous sucrose density gradients, α-MSH_i was found to be associated primarily with subcellular particles which resembled isolated nerve terminals, i.e., synaptosomes. Low to undetectable amounts of α-MSH_i were found in the cytosol or the myelin/microsome fraction of the gradients. The results of these studies are consistent with the view that α-MSH is a neuronal peptide in the human brain.     

Differential effect of ACTH1–24 and α-MSH induced grooming in the paraventricular nucleus of the hypothalamus

Injection of ACTH_1–24 as well as α-MSH in the paraventricular nucleus of the hypothalamus (PVH) induces intense grooming in the rat. While comparing the details of MSH, ACTH and control grooming, we found that the induction of grooming was highly site specific. Even injection of saline in that specific area produced some grooming, possibly due to the release of endogenous substances. To distinguish between effects caused by the peptides and the effects caused by the injection procedure, we compared the behavioural effects of saline and peptide injections in sites with exactly the same location in the PVH, in a post-hoc matched pairs design. Using this design we found that the grooming response induced by saline is of a limited. ACTH_1–24 and α-MSH prolong grooming beyond that period. Interestingly, rats receiving α-MSH continued to groom, while rats receiving ACTH_1–24 changed to scratching. This confirms earlier findings suggesting that grooming and scratching have a differential organization at the level of the PVH. Whether the peptides also have a role in the initiation of the grooming response, or just prolong a response caused by other local factors requires another experimental approach.     

Whole brain and regional [I]-α-bungarotoxin binding in developing rat

These experiments were conducted in order to determine if the total number of binding sites for [¹²⁵I]-α-bungarotoxin ([¹²⁵I]-α-BGT) in rat brain increases and then decreases during postnatal development as predicted by comparison with skeletal muscle, and, if so, to determine at approximately what age the peak in binding occurs in the brain as a whole. A further purpose was to investigate the time-course of development of the [¹²⁵I]-α-BGT binding sites in several brain regions.Specific binding for [¹²⁵I]-α-BGT was studied using the pellets from a 20 min, 14,000 × g centrifugation of rat brain homogenates from 4 or 5 postnatal ages. At least three binding assays were done per region and per age, on cerebral cortex, cerebellum, caudate-putamen, posterior hippocampus, pons-medulla and whole brain. In most regions, the [¹²⁵I]-α-BGT specific binding is measurable, but is low at day one, peaks at about 12–20 days and declines by adulthood. With a few exceptions, these data hold true whether binding is expressed as specific binding per mg protein, specific binding per gram wet tissue, or total specific binding per brain region. The absolute number of specifically bound [¹²⁵I]-α-BGT molecules is undistorted by simultaneous or non-linear growth of cells uninvolved with α-BGT binding and, thus, is the measurement most useful in determining developmental changes. Whole brain has the same age-related pattern as in the majority of the brain regions, i.e., compared to 19–20 days, the adult brain actually has fewer total binding sites.     

ACTH- and α-MSH-induced grooming, stretching, yawning and penile erection in male rats: Site of action in the brain and role of melanocortin receptors

The effect of adrenocorticotropin (ACTH)(1-24) and α-melanocyte stimulating hormone (α-MSH) on grooming, stretching, yawning and penile erection was studied after injection into different brain areas. Both peptides induce the above responses when injected into the hypothalamic periventricular region of the third ventricle. This region includes the paraventricular nucleus, the dorsomedial nucleus, the ventromedial nucleus and the anterior hypothalamic area. The minimal effective dose of both peptides was 0.5 μg and the maximal effect was seen with 2 μg, the highest dose tested. Irrespective of the injection site, grooming started 5–7 min after injection of either peptide, while stretching, yawning and penile erection started only after 15–35 min and lasted for 90–120 min. In contrast both peptides were ineffective when injected into the preoptic area, the caudate nucleus or the CA1 field of the hippocampus. Grooming, stretching and yawning, but not penile erection, were prevented by cyclic[AcCys¹¹, D-Nal¹⁴, Cys¹⁸, AspNH₂²²]-β-MSH (11-22) (HS014), a selective melanocortin 4 receptor antagonist, injected into the same periventricular area 10 min before of ACTH(1-24) or α-MSH. The results show that ACTH(1-24) and α-MSH act in the hypothalamic periventricular region to induce the above responses and that grooming, stretching and yawning, but not penile erection, are mediated by melanocortin 4 receptors.     

Modulation of brain polyphosphoinositide metabolism by ACTH and β-endorphin: Structure-activity studies

This study describes effects of ACTH_1–24 and β-endorphin on brain polyphosphoinositide metabolism in vitro. The interconversion of these polyanionic phospholipids was studied by incubation of a lysed synaptosomal fraction with [γ-³²P]ATP. Of the membrane phospholipids only PA, DPI and TPI became labeled. The reference peptide ACTH_1–24 stimulated the formation of TPI and inhibited the production of PA. For effects on TPI formation both the sequences ACTH_5–7 and ACTH_10–16 were needed. Effects of PA formation required the sequences ACTH_7–10 and ACTH_10–16. The basic amino acids in ACTH_10–16 seemed to be of crucial importance for the peptide effects. A stimulatory effect on DPI was visible when ACTH was shortened from the N-terminus, and the essential information was in ACTH_7–10. β-endorphin inhibited PA formation and this effect was abolished by C-terminal shortening to γ-endorphin. Other fragments of the C-terminus of β-LPH, including the enkephalins, were ineffective. It is concluded that the structure-activity relationship obtTPI/PA formation correlates with a similar relationship obtained on excessive grooming behavior in vivo. A possible correlation between the effects on polyPI metabolism and opiate-like effects, and effects on extinction of active avoidance behavior in vivo is discussed.     

Functional recovery after delayed treatment of ischemic stroke with melanocortins is associated with overexpression of the activity-dependent gene Zif268

Melanocortin peptides afford strong neuroprotection and improve functional recovery in experimental ischemic stroke; they also have established neurotrophic actions. The expression of the immediate early gene Zif268 is dependent on synaptic activity and is involved in injury repair and memory formation. Here, we investigated the role of Zif268 in learning and memory recovery after delayed treatment of ischemic stroke with the melanocortin analog [Nle⁴, d-Phe⁷]α-MSH (NDP-α-MSH). A 10-min period of global cerebral ischemia was induced by occluding both common carotid arteries in gerbils. Treatment with a nanomolar dose of NDP-α-MSH (every 12 h for 11 days) was performed starting 3 h or 9 h after stroke induction; where indicated, gerbils were pretreated with the melanocortin MC₄ receptor antagonist HS024. Animals were subjected to the Morris water-maze test (four sessions from 4 to 50 days after the ischemic episode). Fifty days after stroke, histological damage and Zif268 expression were investigated in the hippocampus. Treatment with NDP-α-MSH significantly reduced hippocampal damage, including neuronal death, and improved learning and memory recovery. This protective effect was long-lasting (50 days, at least) and associated with Zif268 overexpression, with both schedules of NDP-α-MSH treatment. Pharmacological blockade of MC₄ receptors prevented these effects. Our data indicate that MC₄ receptor-mediated actions of melanocortins could trigger repair mechanisms able to improve neuronal functionality and synaptic plasticity, and to promote long-lasting functional recovery from ischemic stroke with Zif268 gene involvement.     

The influence of ACTH and corticosterone on [H]GABA receptor binding in rat brain

Bilateral adrenalectomy (ADX) induces a significant, regionally selective, increase in GABA, but not cholinergic muscarinic orα₁-adrenergic, receptor binding in rat brain. The increase in GABA receptor binding in the midbrain occurs within 72 h of surgery, whereas that found in the corpus striatum becomes evident between 1 and 2 weeks later. These ADX-induced receptor changes are counteracted by the administration of corticosterone, a reversal which can occur within 24 h following a single administration of the steroid. Unlike ADX, hypophysectomy causes a significant reduction in [³H]GABA receptor binding in these two brain areas, an action that is not reversed by corticosterone treatment. Furthermore, systemic administration of either ACTH_1–39 or ACTH_4–10 in unoperated animals causes an increase in midbrain and striatal GABA receptor binding similar to that observed in ADX animals. The increase in [³H]GABA binding observed after ACTH administration appears to be due to the apearance of low affinity, high capacity binding sites not observed in untreated animals. ADX had no effect on high affinity GABA uptake, glutamic acid decar☐ylase or GABA content in the brain regions where receptor modifications were noted. These findings indicate that GABA receptor binding in rat brain can be modified by changes in the circulating levels of ACTH.     

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.     

Characterization of [I]HEAT binding to α1-receptors in human brain: assessment in aging and Alzheimer's disease

The binding of [¹²⁵I]2-(β-4-hydroxyphenylethylamino-ethyltetralone ([¹²⁵I]HEAT), an α₁-adrenergic receptor antagonist, to human brain membranes was characterized and the binding assessed in tissue from subjects with Alzheimer's disease (AD) and aging controls. Under Na⁺-K⁺ phosphate buffer conditions, [¹²⁵I]HEAT bound to a single class of binding sites in prefrontal cortex (Brodmann area 10) with a K_d of about 120 pM. High binding capacities of [¹²⁵I]HEAT were evident in the hippocampus and neocortex but were low in subcortical areas and cerebral microvessels comparable to the regional distribution of [³H]prazosin binding reported previously. Displacement of [¹²⁵I]HEAT by various adrenergic drugs was consistent with its binding to α₁-adrenergic receptors. The specific binding was not affected by postmortem delay between death and freezing of tissue at autopsy. There was no correlation of [¹²⁵I]HEAT binding with age of subjects. In AD subjects, the binding was significantly decreased in prefrontal cortex by about 25% but not changed in hippocampus, putamen or cerebellum compared to age-matched controls. The reduced binding of [¹²⁵I]HEAT in prefrontal cortex may reflect a region-specific change in α₁-adrenergic receptors associated with neuronal loss in AD.     

Axonal transport of α-bungarotoxin binding sites in rat sciatic nerve

[¹²⁵I]α-Bungarotoxin (α-BuTX) binding sites accumulate both proximal and distal to a ligature positioned around the sciatic nerve of rats. [¹²⁵I]α-BuTX binding sites, localized using quantitative receptor autoradiography, were found to accumulate at nerve ligatures at a relatively constant rate which suggests that they undergo both anterograde and retrograde axonal transport. [¹²⁵I]α-BuTX binding to sections of ligated sciatic nerve was saturable with apparent dissociation constants of 0.97 nM proximal and 0.53 nM distal to the ligature.d-Tubocurarine, nicotine, decamethonium and atropine displaced [¹²⁵I]α-BuTX from sciatic nerve sections with affinities comparable to those previously reported for the toxin binding component of rat brain. These data indicate that [¹²⁵I]α-BuTX binding sites pharmacologically similar to those of rat brain are transported in sciatic nerve. Axonally transported toxin binding sites may correspond to those previously localized to the plasma membrane of peripheral nerve axons and on the terminals of motor neurons.     

Modulation of Interleukin-1 Receptors in the Brain-Endocrine-Immune Axis by Stress and Infection

We summarize data from some of our recent studies on in vitro and in vivo modulation of interleukin-1 (IL-1) receptors in the mouse brain-endocrine-immune axis by stress and infection. Ether-laparotomy stress in mice resulted in a selective increase in pituitary IL-I receptors and a significant decrease in pituitary receptors for corticotropin-releasing factor (CRF), a major regulator of the endocrine response to stress. Intraperitoneal injection of rat/human CRF mimicked the effects of stress and resulted in a dramatic increase in [¹²⁵I]IL-1α binding in the pituitary; [¹²⁵I]IL-1α binding in the hippocampus, spleen, and testis was unaffected by stress or CRF treatment. Glucocorticoid treatment with dexamethasone alone did not alter [¹²⁵I]IL-1α, binding but significantly inhibited CRF-induced upregulation of IL-1 receptors in the pituitary. The intracellular mechanism(s) involved in stress and CRF-induced upregulation of IL-1 receptors in the pituitary gland were examined by evaluating the effects of treatment of AtT-20 mouse pituitary corticotroph cells with a variety of neuroendocrine mediators of stress. CRF, forskolin, and isoproterenol (β₂ adrenergic receptor agonist) produced dose-dependent increases in cAMP production and [¹²⁵I]IL-1α binding. In contrast, somatostatin and dexamethasone significantly inhibited CRF-stimulated increase of cAMP production and [¹²⁵I]IL-1α binding, suggesting a primary role for cAMP in the regulation of pituitary IL-1 receptors. Next, we investigated the modulation of LL-1 β levels and IL-1 receptors following infection of mice with the endotoxin, lipopolysaccharide (LPS). Acute administration of low doses of endotoxin (30 μg LPS/mouse) dramatically increased IL-1 β levels and reciprocally decreased [¹²⁵I]IL-1α binding in peripheral tissues (pituitary, testis, liver, and spleen) but not in brain (hippocampus). This effect appeared to be dose related since higher doses of endotoxin (300 μg LPS/mouse) significantly decreased [¹²⁵I]IL-1α binding in both peripheral tissues and brain. Endotoxin induced modulation of the IL-1 system was also dependent on the treatment regimen since two low-dose LPS injections (at 0 and 12 h) increased IL-1 β concentrations and decreased [¹²⁵I]IL-1α binding in both central and peripheral tissues. These data provide further support for a role for IL-1 in coordinating brain-endocrine-immunoresponses to stress and infection.     

α-Bungarotoxin binding to a high molecular weight component from lower vertebrate brain identified on dodecyl sulfate protein-blots

The binding of [¹²⁵I]iodo-α-bungarotoxin ([¹²⁵]α-BuTX) to the dissociated α-subunit of Torpedo acetylcholine receptor (AChR) can be readily demonstrated in a modified ‘protein-blot’ analysis utilizing electrophoretically transferred, dissociated subunits immobilized onto positively charged nylon membranes which are then incubated directly with [¹²⁵I]α-BuTX. We report here the use of the protein-blotting technique to detect the α-BuTX binding site present in the central nervous system of lower vertebrates and to characterize some of the physicochemical properties of the toxin binding site. High molecular weight (M200,000 and 120,000) α-BuTX-binding components can be readily demonstrated in avian and fish brain extracts upon protein-blotting with [¹²⁵I]α-BuTX following lithium dodecyl sulfate PAGE. Neither extensive reduction with dithiothreitol nor prior reduction followed by alkylation with iodoacetamide alter the mobility of the CNS-derived BuTX-binding sites. In contrast to our findings with Torpedo AChR or muscle AChR derived from a number of different species, no binding is observed in the molecular weight range of the α-subunit (M_r= 40,000) nor is any binding at any molecular weight observed in similar fractions prepared from adult, mammalian (rat, guinea pig) brain using this technique. These results demonstrated the existence in lower vertebrate brain of a BuTX binding site comparable in size to the AChR oligomeric complex of electric organ and muscle. They also suggest, however, striking structural differences between muscle AChR and the central neuronal BuTX-binding complex as well as a considerable difference between the neuronal BuTX-binding sites derived from lower and higher vertebrate brain.     

α-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with α7 subunits and up-regulation by chronic nicotine treatment

High density neuronal cultures from rat E18 hippocampus and cortex have been characterised with respect to cholinergic binding sites. No specific binding of [³H]nicotine or [³H]cyttine to live cells in situ was detected, although the limit for detection was estimated to be 30 fmol/mg protein. Muscarinic binding sites labelled with [³H]QNB were present at a density of 0.75 pmol/mg protein. [¹²⁵I]α-Bungarotoxin (αBgt) bound to hippocampal cultures with a B_max of 128 fmol/mg protein and a K_d of 0.6 nM; cortical cultures expressed five times fewer [¹²⁵I]α-Bgt binding sites. Fluorescence cytochemistry with rhodamine-α-Bgt indicated that 95% of hippocampal neurons were labelled, compared with only 36% of cortical neurons. Average densities of 4 × 10⁴ and 2 × 10⁴ binding sites/cell were calculated for hippocampal and cortical cultures, respectively. Double labelling experiments with mAb307 (which recognises the rat α7 nicotinic receptor subunit) and rhodamine-α-Bgt gave coincident labelling patterns, supporting the correlation between the α7 subunit and Bgt-sensitive neuronal nicotinic receptor. Treatment of hippocampal cultures with 10 μM nicotine for 14 days elicited a 40% increase in the numbers of [¹²⁵I]α-Bgt binding sites, mimicking the up-regulation observed in vivo studies. Primary cultures offer a useful in in vitro system for investigating the expression and regulation of brain α-Bgt-sensitive receptors.     

Effects of gonadal steroids on the in vivo binding of [I]α-bungarotoxin to the suprachiasmatic nucleus

The radioligand [¹²⁵I]α-bungarotoxin (α-BTX) has been used to test receptor binding to putative nicotinic cholinergic receptors in the hypothalamus. Using light microscopic autoradiography following third ventricular infusion of the radioligand we have previously demonstrated that in normally cycling rats and in normal males, the suprachiasmatic nucleus (SCN) consistently binds the α-neurotoxin. In chronically (5 weeks) oophorectomized female, binding of [¹²⁵I]α-BTX to the SCN is markedly diminished. The present series of experiments were designed to test the effects of gonadal steroids on the binding of [¹²⁵I]α-BTX to the SCN. We first tested whether or not estradiol administered to ovariectomized females could duplicate the presence of the ovary. In females ovariectomized and immediately provided with a constant dose of estradiol-17β (E₂)—1.0 cm silastic capsules for 5 weeks, (n= 4), the binding of the neurotoxin to the SCN was maintained. In females ovariectomized for 3 weeks and replaced with E₂ for 2 weeks (n= 4), the binding of [¹²⁵I]α-BTX to the SCN was restored. In chronically (4 weeks) ovariectomized females receivingf E₂ for 6 dyas (n= 2), the binding of the neurotoxin was partially restored. We next tested the effect of chronic (5 weeks) castration (n= 100) and observed that binding of [¹²⁵I]α-BTX to the SCN of castrated males was like that of intact male controls (n= 6). We also tested the effect of neonatal adrogenization (1.25 mg testosterone proprionate, neonatal days 2 and 5) with subsequent adult oophorectomy (day 83) and observed that neonatally androgenized females with intact ovaries (n= 4) demonstrated [¹²⁵I]α-BTX binding to the SCN like that of non-adrogenized females, while neonatally androgenized females without ovaries (n= 4) had decreased binding of the neurotoxin to the SCN like that of oophorectomized adult females. These data demonstrate that: (1) the administration of estradiol can maintain and restore [¹²⁵I]α-BTX labeling of the SCN in ovariectomized rats; (2) binding of [¹²⁵I]α-BTX to the SCN is different in castrated males and ovariectomized females; and (3) the difference in α-BTX binding to the SCN receptor in adult castrated male and female rats may be dependent upon the presence of gonadal steroids at times other than the ‘critical’ postnatal period.     

A radioiodinated 7 α -O-iodoallyl diprenorphine for mapping opioid receptors

The aim of the current research has been to validate an original radioiodinated diprenorphine (DPN) derivative suitable for imaging studies of opioid receptors. [¹²⁵I]7α-O-iodoallyl diprenorphine (7α-O-IA-DPN) was prepared by radioiododestannylation and in vitro and in vivo opioid receptor binding assays were performed with CDF1 mouse brains.In vitro binding studies showed high affinity (K_i= 0.4 ± 0.2 nM) for mouse brain membranes. In vivo studies showed 63% specific binding. Ex vivo autoradiography of brain sections confirmed high uptake and retention of [¹²⁵I]7α- O -IA-DPN in regions rich in opioid receptors. This new radioiodinated DPN analogue appears to be a potential radioprobe for in vivo visualization of human cerebral opioid receptors with single photon emission computed tomography (SPECT).     

α1-Adrenergic receptors and stimulation of [H]inositol metabolism in rat brain: Regional distribution and parallel inactivation

The relationship between norepinephrine-stimulated phosphatidyl-inositol metabolism andα₁-adrenergic receptor density was examined in rat brain. Increases in phosphatidyl-inositol metabolism were determined by accumulation of [³H]inositol phosphates in the presence of lithium in brain slices, while receptor density was determined by specific binding of¹²⁵I-BE 2254 (¹²⁵IBE) in membrane fractions. Treatment of slices of cerebral cortex with increasing concentrations of the irreversibleα₁-adrenergic receptor antagonist phenoxybenzamine caused a parallel inactivation of specific¹²⁵IBE binding sites and norepinephrine-induced [³H]inositol phosphate accumulation, although approximately 20% of the binding sites remained after abolition of the inositol response. Comparison of the density of¹²⁵IBE binding sites and the magnitude of norepinephrine-stimulated [³H]inositol phosphate accumulation in 8 different brain regions did not show a particularly good correlation. The thalamus had the highest density of binding sites and an intermediate inositol response, while the hippocampus had the highest inositol response but an intermediate density of binding sites. However, the cerebellum had the lowest density of binding sites and no measurable inositol response. Treatment of slices of each region with 300 nM phenoxybenzamine abolished the inositol response and caused a 59–73% decrease in the density of¹²⁵IBE binding sites. The lack of correlation between receptor density and inositol response between brain regions could not be explained on the basis of receptor affinity, spare receptors, protein content, nor differences in slice size. These results suggest thatα₁-adrenergic receptors labeled by¹²⁵IBE are coupled to [³H]inositol metabolism in rat brain, but the receptor density is not the sole determinant of the magnitude of norepinephrine-induced increases in [^3H]inositol metabolism.     

Contrasting effect of a brain supernatant extract on neuronal vs neuromuscular α-bungarotoxin receptors

The effects of a rat brain supernatant extract and a partially purified supernatant preparation from bovine brain were determined on the binding of [¹²⁵I]α-bungarotoxin (α-BGT) to muscle membranes, as well as to membranes prepared from brain. In agreement with previous work, the supernatant preparations inhibited α-BGT binding to brain membranes in a dose-dependent fashion, (Brain Research, 245 (1982) 57–67); however, no significant effect of either of the preparations was observed on the binding of the toxin to muscle membranes. As well, the supernatant preparations did not affect binding of radiolabelled α-BGT to muscle cells in culture in competition binding experiments. The effect of long-term incubation of cells in culture with the supernatant preparations was subsequently determined. These studies showed that the binding of [¹²⁵I]α-BGT increased markedly (300%) in the presence of a crude rat brain supernatant preparation, while incubation of the muscle cells in the presence of the partially purified bovine supernatant extract had no significant effect on radiolabelled toxin binding. In contrast, both the rat and bovine supernatant preparations significantly decreased (up to 65%) radiolabelled toxin binding to a cultured neuronal cell population, adrenal medullary chromaffin cells. These results suggest that an endogenous factor(s), present in brain extracts, differentially regulates the neuronal as compared to the neuromuscular nicotinic α-bungarotoxin binding sites.     

[I]4-Aminobenzyl-5′-N-methylcarboxamidoadenosine ([I]AB-MECA) labels multiple adenosine receptor subtypes in rat brain

Adenosine modulates neuronal activity and neurotransmitter release through interaction with cell surface receptors. Four adenosine receptor subtypes, A₁, A_2A, A_2B, and A₃ receptors, have been cloned and characterized. The agonist ligand, [¹²⁵I]AB-MECA ([¹²⁵I]4-aminobenzyl-5′-N-methylcarboxamidoadenosine) has high affinity for recombinant A₁ and A₃ receptors [Olah et al., Mol. Pharmacol., 45 (1994) 978–982]. Rodent A₃ receptors are relatively insensitive to xanthines; inhibition of A₁ receptors with xanthines allows selective detection of A₃ receptors despite the lack of selectivity of the ligand. We studied whether [¹²⁵I]AB-MECA is useful for localization and characterization of A₃ receptors in rat brain. The autoradiographic distribution of total [¹²⁵I]AB-MECA (400 pM) binding closely resembled the pattern of A₁ receptor binding, with highest levels in cerebellum, hippocampus, and thalamus, and moderate levels in cortex and striatum. Drug competition studies confirmed that almost all [¹²⁵I]AB-MECA binding could be attributed to labeling of A₁ receptors. Xanthine amine congener (1 μM) reduced specific [¹²⁵I]AB-MECA binding by >95%, indicating that xanthine-resistant A₃ receptors represent a quantitatively minor subtype. Despite the use of a radioligand with high affinity and high specific activity, the low density of A₃ receptors in rat brain appears insufficient to allow localization, or even consistent detection, of this receptor subtype. In the presence of DPCPX (50 nM, to block A₁ receptors), residual [¹²⁵I]AB-MECA binding to A_2A receptors was observed in the striatum. Thus, [¹²⁵I]AB-MECA labels primarily A₁ and A_2A adenosine receptors in rat brain.