Galanin receptors and their second messengers in the lumbar dorsal spinal cord

The ligand binding properties of galanin receptors were examined in crude synaptosomal fraction preparations of lumbar dorsal spinal cord, using chloramin-T mono-iodinated porcine Tyr²⁶ galanin as ligand. The equilibrium binding of [¹²⁵I]galanin showed the presence of a single population of high-affinity binding sites with K_D= 0.6±0.2 nM in a concentration of 55±15 fmol mg^-1 protein (B_max. The N-terminal fragments galanin (1–16) and galanin (1–12) fully displaced specific [¹²⁵1]galanin binding from membranes with IC₅₀ values 6 nM and 4 μM, respectively. The C-terminal fragment galanin (17–29) did not displace [¹²⁵I]galanin when applied in the concentration range 10^-11–10^-4 M. GTP inhibited the specific binding of [¹²⁵I] galanin in a concentration dependent manner, with 54% inhibition at 1 mM, suggesting that the galanin receptor found in lumbar dorsal spinal cord is G-protein coupled. Second messenger systems, through which the galanin receptor in lumbar dorsal spinal cord may exert its effect, were also studied. A galanin (10,μm) produced inhibition (58%) of the depolarization induced cGMP increase was found, whereas galanin (10 μM) did not inhibit the noradrenalin (100 μM) activated CAMP synthesis or phosphoinositide turnover in tissue slices of the spinal cord. Bilateral transection of the sciatic nerve at midthigh level 14 days prior to the binding experiment was performed, a treatment which is known to cause a dramatic increase of galanin-like immunoreactivity in the superficial layers of the dorsal spinal cord, dorsal root ganglion and in galanin mRNA levels, but no significant effect on B_max or K_D of the galanin receptor was found.     

Transferrin receptors on the plasma membrane of cultured rat astrocytes

It is generally accepted that transferrin receptor is located in the endothelial cells of the brain, but its existence in other brain cell types is less established. In this study, a [¹²⁵I]transferrin binding assay was used to determine whether there is transferrin receptor on the membrane of cultured rat cortical astrocytes (type 1) in vitro. The results demonstrated that cortical astrocytes (type 1) in suspension attracted [¹²⁵I]transferrin with a saturable and specific binding. Scatchard and Hill plot analysis showed that the dissociation constant (K_D) of the binding was about 3.5×10^–8 M and the number of receptors was about 7.1×10⁴/cell. The Hill coefficient was 0.99, approaching 1, indicating the absence of cooperativity. The receptor was specific both for rat and human transferrin. The binding of rat [¹²⁵I]transferrin could be competitively and specifically inhibited by unlabeled iron-saturated rat and human transferrin, and no difference was found between interaction of rat or human transferrin with this receptor. The interaction of duck or camel transferrin with this receptor was found to be very weak. This study provides evidence for the presence of transferrin receptor on the plasma membrane of cultured rat brain astrocytes. Received: 25 May 1999 / Accepted: 18 August 1999     

Insulin receptors along the rat nephron: [125I] Insulin binding in microdissected glomeruli and tubules

Binding of [¹²⁵I] Tyr A¹⁴ human insulin ([¹²⁵I] insulin) was measured at 4°C in glomeruli and pieces of tubule microdissected from collagenase-treated rat kidneys. For glomeruli and all segments tested, total and non specific binding increased linearly with glomeruli number or tubular length. When determined with 4.0 nM labelled hormone, the distribution of specific binding sites (expressed as 10^–18 mol [¹²⁵I] insulin bound per glomerulus or mm tubule length) was as follows: glomerulus, 2.5±0.3; proximal convoluted tubule (PCT), 12.6±0.6; pars recta (PR), 4.0±2.3; thin descending limb (TDL), 0.6±0.2; thin ascending limb (TAL), 0.6±0.2; medullary thick ascending limb (MAL), 0.8±0.1; cortical ascending limb (CAL), 2.1±0.1; distal convoluted tubule (DCT), 5.6±1.1; cortical collecting tubule (CCT), 3.2±0.3 and outer medullary collecting tubule (MCT), 2.3±0.1. Specific [¹²⁵I] insulin binding to glomeruli and tubule segments was time- and dose-dependent, saturable, reversible after elimination of free labelled ligand, and inhibited by unlabelled human insulin. When analysed in Scatchard and Hill coordinates, the binding data revealed a negative cooperation in the interaction processes between [¹²⁵I] insulin and glomerular and tubular binding sites, with apparent dissociation constants and Hill coefficients of the following values: glomerulus, 0.6 nM and 0.60; PCT, 10.0 nM and 0.55; MAL, 4.3 nM and 0.80; CAL, 2.0 nM and 0.74; CCT, 7.6 nM and 0.80 and MCT, 1.0 nM and 0.57 respectively. The stereospecificity of nephron binding sites was assessed in competitive experiments showing that unlabelled bovine and procine insulins were as efficient as human insulin for displacing [¹²⁵I] insulin, whereas A and B chains of insulin and unrelated peptide hormones were almost inactive. These results indicate that the detected [¹²⁵I] insulin binding sites may correspond to physiological insulin receptors.Abbreviations used [¹²⁵I] Insulin [¹²⁵I] Tyr A¹⁴ human insulin - PCT proximal convoluted tubule - PR pars recta - TDL thin descending limb - TAL thin ascending limb - MAL medullary thick ascending limb - CAL cortical ascending limb - DCT distal convoluted tubule - CCT cortical collecting tubule - MCT outer medullary collecting tubule     

Comparative laminar distribution of various autoradiographic cholinergic markers in adult rat main olfactory bulb

To provide anatomical information on the complex effects of acetylcholine (ACh) in the olfactory bulb (OB), the distribution of different cholinergic muscarinic and nicotinic receptor sub-types was studied by quantitative in vitro autoradiography. The muscarinic M1-like and M2-like sub-types, as well as the nicotinic bungarotoxin-insensitive (α4β2-like) and bungarotoxin-sensitive (α7-like) receptors were visualized using [³H]pirenzepine, [3H]AF-DX 384, [³H]cytisine and [¹²⁵I]α-bungarotoxin (BTX), respectively. In parallel, labelling patterns of [³H]vesamicol (vesicular acetylcholine transport sites) and [³H]hemicholinium-3 (high-affinity choline uptake sites), two putative markers of cholinergic nerve terminals, were investigated. Specific labelling for each cholinergic radioligand is distributed according to a characteristic laminar and regional pattern within the OB revealing the lack of a clear overlap between cholinergic afferents and receptors. The presynaptic markers, [³H]vesamicol and [³H]hemicholinium-3, demonstrated similar laminar pattern of distribution with two strongly labelled bands corresponding to the glomerular layer and the area around the mitral cell layer. Muscarinic M1-like and M2-like receptor sub-types exhibited unique distribution with their highest levels seen in the external plexiform layer (EPL). Intermediate M1-like and M2-like binding densities were found throughout the deeper bulbar layers. In the glomerular layer, the levels of muscarinic receptor subtypes were low, the level of M2-like sites being higher than M1. Both types of nicotinic receptor sub-types displayed distinct distribution pattern. Whereas [¹²⁵I]α-BTX binding sites were mostly concentrated in the superficial bulbar layers, [³H]cytisine binding was found in the glomerular layer, as well as the mitral cell layer and the underlying laminae. An interesting feature of the present study is the visualization of two distinct cholinoceptive glomerular subsets in the posterior OB. The first one exhibited high levels of both [³H]vesamicol and [³H]hemicholinium-3 sites. It corresponds to the previously identified atypical glomeruli and apparently failed to express any of the cholinergic receptors under study. In contrast, the second subset of glomeruli is not enriched with cholinergic nerve terminal markers but displayed high amounts of [³H]cytisine/nicotinic binding sites. Taken together, these results suggest that although muscarinic receptors have been hypothesized to be mostly involved in cholinergic olfactory processing and short-term memory in the OB, nicotinic receptors, especially of the cytisine/α4β2 sub-type, may have important roles in mediating olfactory transmission of efferent neurons as well as in a subset of olfactory glomeruli.     

Autoradiographic quantitation and anatomical mapping of GTP sensitive-galanin receptors in the guinea pig central nervous system

Galanin is a 29-amino acid peptide widely distributed in the mammalian central nervous system. Galanin receptors in the guinea pig brain were visualized using [¹²⁵I]galanin by in vitro receptor quantitative autoradiography. Scatchard analysis of [¹²⁵I]galanin binding to slide-mounted sections revealed saturable binding to a single class of high affinity receptors with a K_D of approximately 1 nM. Specific [¹²⁵I]galanin binding sites were detected in a large number of brain areas (concentration range: from non detectable to 99.32 fmol/mg of tissular proteins). The anatomical mapping revealed high densities essentially in the telencephalon (e.g. lateral septal nuclei, amygdala, hippocampal dentate gyrus) and the diencephalon (e.g. the anterodorsal and medial habenular thalamic nuclei, the paraventricular, dorsomedian and median mammillary hypothalamic nuclei, the posterior lobe of the pituitary). Addition of Mg²⁺ and GTP increased binding in some areas such as the zona incerta, the median eminence and the arcuate nucleus, and decreased it in other areas such as the amygdala, the hippocampus and the mammillary nuclei. This regional heterogeneity in the effect of Mg²⁺ and GTP can be interpreted as: (1) different rates of galanin receptor occupancy by endogenous peptide; (2) a differential coupling of GTP binding proteins to galanin receptors in the brain structures; and (3) a different nature of receptors. At any rate, this study provides evidence for a specific GTP-sensitive galanin receptor in guinea pig brain with an extensive distribution suggesting various physiological implications. Comparison with studies performed in several mammals shows that the overall distribution of galanin receptors is well preserved among species. These data suggest that galanin may posses similar functional properties in the different species tested so far. Nevertheless, very distinct differences were found in some areas like the cortex, the hippocampus and the pituitary.     

Glucagon receptors along the nephron: [125I]glucagon binding in rat tubules

Binding of [¹²⁵I]glucagon was measured in microdissected pieces of tubules from the rat nephron. Specific glucagon binding sites were found only in nephron segments containing a glucagon-sensititive adenylate cyclase activity. At 7.5 nM labelled hormone, higher levels of specific binding (16–27×10^–18 mol mm^–1) were found in the thick ascending limb of the Henle's loop and in the distal convoluted tubule and lower binding levels (2–5×10^–18 mol mm^–) in the collecting tubule whereas specific binding could not be detected in the proximal tubule and in the thin segments of the Henle's loop. In the medullary thick ascending limb, Scatchard analysis of specific [¹²⁵I]glucagon binding indicated an apparent equilibrium dissociation constant of 2.4 nM. The stereospecificity of binding sites in medullary thick ascending limbs and medullary collecting tubules, was assessed by competition experiments using unlabelled glucagon, enteroglucagon and unrelated hormones (vasopressin, calcitonin, parathyroid hormone and insulin); in both segments, glucagon was more active than enteroglucagon in displacing labelled glucagon from its tubular binding sites, whereas all other hormones tested were inactive. These results indicate that tubule binding sites might be the physiological receptors for glucagon involved in adenylate cyclase activation.Abbreviations PCT proximal convoluted tubule - TDL thin descending limb - TAL thin ascending limb - MAL medullary thick ascending limb - CAL cortical ascending limb - DCT distal convoluted tubule - CCT cortical collecting tubule - MCT medullary collecting tubule     

D1-like dopamine receptors on retrogradely labelled sympathoadrenal neurones in the thoracic spinal cord of the rat

Cellular localization of dopamine D₁-like receptors was accomplished on target-specified sympathoadrenal preganglionic neurones using the radioligand [³H]SCH23390. Sympathoadrenal neurones were retrogradely labelled with cholera B subunit conjugated to horseradish peroxidase and were detected in segments T₁ to T₁₃ with a predominance at T₈/T₉. Binding of the selective D₁-like radioligand [³H]SCH23390 was associated with the retrogradely labelled sympathoadrenal neurones in longitudinal/horizontal sections of thoracic spinal cord. D₁-like receptor localization on target-specific neurones was determined in more than half of the spinal cord sections and was associated predominantly with the cell soma and principal proximal dendrites in the intermediolateral cell column of the spinal grey matter. D₂-like receptor localization was not associated with retrogradely labelled sympathoadrenal neurones but a higher degree of specific binding was noted in more medial aspects of the spinal grey matter. This is the first successful demonstration of receptor localization combining two quite different techniques and provides conclusive anatomical evidence for D₁-like receptor localization on sympathetic preganglionic neurones that project to the adrenal medulla. Received: 27 November 1998 / Accepted: 6 May 1999     

Galanin receptors and their second messengers in the lumbar dorsal spinal cord.

The ligand binding properties of galanin receptors were examined in crude synaptosomal fraction preparations of lumbar dorsal spinal cord, using chloramin-T mono-iodinated porcine Tyr26 galanin as ligand. The equilibrium binding of [125I]galanin showed the presence of a single population of high-affinity binding sites with KD = 0.6 +/- 0.2 nM in a concentration of 55 +/- 15 fmol mg-1 protein (Bmax). The N-terminal fragments galanin (1-16) and galanin (1-12) fully displaced specific [125I]galanin binding from membranes with IC50 values 6 nM and 4 microM, respectively. The C-terminal fragment galanin (17-29) did not displace [125I]galanin when applied in the concentration range 10(-11)-10(-4) M. GTP inhibited the specific binding of [125I] galanin in a concentration dependent manner, with 54% inhibition at 1 mM, suggesting that the galanin receptor found in lumbar dorsal spinal cord is G-protein coupled. Second messenger systems, through which the galanin receptor in lumbar dorsal spinal cord may exert its effect, were also studied. A galanin (10 microM) produced inhibition (58%) of the depolarization induced cGMP increase was found, whereas galanin (10 microM) did not inhibit the noradrenalin (100 microM) activated cAMP synthesis or phosphoinositide turnover in tissue slices of the spinal cord. Bilateral transection of the sciatic nerve at midthigh level 14 days prior to the binding experiment was performed, a treatment which is known to cause a dramatic increase of galanin-like immunoreactivity in the superficial layers of the dorsal spinal cord, dorsal root ganglion and in galanin mRNA levels, but no significant effect on Bmax or KD of the galanin receptor was found.     

Autoradiographic localization of binding sites for arginine vasopressin and atrial natriuretic peptide on astrocytes and neurons of cultured rat central nervous system.

The cellular localization of binding sites for [125I]arginine vasopressin and [125I]atrial natriuretic peptide was studied in explant cultures of rat spinal cord, brain stem and cerebellum by means of autoradiography. In brain stem cultures, especially in the nucleus of the solitary tract, a great number of neurons revealed binding sites for both peptides. In spinal cord cultures, many neurons of various sizes were labelled by [125I]arginine vasopressin, whereas only a small number of cells showed binding sites for [125I]atrial natriuretic peptide. Neurons in cerebellar cultures revealed little or no binding for the peptides. In addition to neurons, binding sites for [125I]arginine vasopressin and [125I]atrial natriuretic peptide were also observed on glial cells. Simultaneous staining of the cultures with glial fibrillary acidic protein has shown that the labelled cells were glial fibrillary acidic protein-positive and could therefore be identified as astrocytes. Labelling of the cells by [125I]arginine vasopressin and [125I]atrial natriuretic peptide was more intense in spinal cord and brain stem cultures than in cultures of cerebellum, providing evidence for a heterogeneity of astrocytes in different regions of the central nervous system. Binding of both [125I]arginine vasopressin and [125I]atrial natriuretic peptide to neurons and astrocytes could be competed by the unlabelled peptides, suggesting specific binding of the radioligands. Our autoradiographic studies provide good evidence that in addition to neurons, astrocytes also express receptors for arginine vasopressin and atrial natriuretic peptide.     

Muscarinic receptors in basal ganglia in dementia with Lewy bodies,Parkinson's disease and Alzheimer's disease

Derivatives of the muscarinic antagonist 3-quinuclidinyl-4-iodobenzilate (QNB), particularly [123I]-(R,R)-I-QNB, are currently being assessed as in vivo ligands to monitor muscarinic receptors in Alzheimer's disease (AD) and dementia with Lewy bodies (DLB), relating changes to disease symptoms and to treatment response with cholinergic medication. To assist in the evaluation of in vivo binding, muscarinic receptor density in post-mortem human brain was measured by autoradiography with [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB and compared to M1 ([3H]pirenzepine) and M2 and M4 ([3H]AF-DX 384) receptor binding. Binding was calculated in tissue containing striatum, globus pallidus (GPe), claustrum, and cingulate and insula cortex, in cases of AD, DLB, Parkinson's disease (PD) and normal elderly controls. Pirenzepine, AF-DX 384 and (R,S)-I-QNB binding in the striatum correlated positively with increased Alzheimer-type pathology, and AF-DX 384 and (R,R)-I-QNB cortical binding correlated positively with increased Lewy body (LB) pathology; however, striatal pirenzepine binding correlated negatively with cortical LB pathology. M1 receptors were significantly reduced in striatum in DLB compared to AD, PD, and controls and there was a significant correlation between M1 and dopamine D2 receptor densities. [3H]AF-DX 384 binding was higher in the striatum and GPe in AD. Binding of [125I]-(R,R)-I-QNB, which may reflect increased muscarinic M4 receptors, was higher in cortex and claustrum in DLB and AD. [125I]-(R,S)-I-QNB binding was higher in the GPe in AD. Low M1 and D2 receptors in DLB imply altered regulation of the striatal projection neurons which express these receptors. Low density of striatal M1 receptors may relate to the extent of movement disorder in DLB, and to a reduced risk of parkinsonism with acetylcholinesterase inhibition.     

Electron microscopic localization of [125I]alpha-bungarotoxin binding sites in the outer plexiform layer of the goldfish retina.

Summary Light and electron microscope autoradiography were performed on goldfish (Carassius auratus) retinas incubated in [¹²⁵I] labelled -bungarotoxin. The toxin was bound preferentially to membrane receptors in the inner and outer plexiform layers. Binding was suppressed by 10^–5 M nicotine or 10^–5 M native -bungarotoxin. Electron microscopic analysis of the outer plexiform layer (OPL) strongly suggested that -bungarotoxin binding sites were located on small bipolar cell dendritic processes that invaginated rod and cone synaptic terminals, and on large bipolar cell dendritic processes more proximally situated in the OPL. Large horizontal cell processes in the OPL and horizontal cell processes that invaginated rod and cone synaptic terminals did not appear to be labelled.     

Characteristics of albumin binding to opossum kidney cells and identification of potential receptors

 Albumin re-absorption in the kidney proximal tubule may be pathophysiological in disease. Opossum kidney (OK) cell monolayers were used to investigate the characteristics of [¹²⁵I]-labelled albumin binding at 4°C. Two binding sites were identified, one with high affinity (K _D 154.8 ±7 mg/l) and low capacity, the other with low affinity (K _D 8300 ± 1000 mg/l) and high capacity. Binding was sensitive to lectins Glycine max and Ulex europaeus I, but not other lectins, indicating involvement of a glycoprotein(s) in the binding process. Binding was also sensitive to a number of agents known to inhibit binding to scavenger receptors. [¹²⁵I]-Labelled albumin ligand blotting of OK cell membrane proteins identified several albumin-binding proteins with identical lectin affinities to those proteins mediating albumin binding to OK cell monolayers. These results provide initial evidence of the identity of albumin receptors in kidney tubules, and suggest that they may be members of the family of scavenger receptors. Received: 24 July 1996 / Received after revision: 16 October 1996 / Accepted: 18 October 1996     

Regional stimulatory and inhibitory effects of guanine nucleotides on [125I]galanin binding in rat brain: relationship with the rate of occupancy of galanin receptors by endogenous galanin.

Galanin has been shown to stimulate feeding or modulate neuroendocrine secretions when administered centrally. In the present work, using quantitative autoradiography, we documented the existence of [125I]galanin specific binding sites in several hypothalamic nuclei expected to mediate these effects. In standard binding conditions, [125I]galanin specific binding can be visualized in the hypothalamic ventromedial nucleus, stria terminalis, piriform cortex, central amygdaloid nucleus and medial amygdaloid nucleus, while it is almost undetectable in most neuroendocrine or autonomic hypothalamic areas. We hypothesized that high endogenous galanin levels in these regions might mask galanin receptors. We first showed that a high ionic strength/acid wash of brain slices is effective in removing more than 80% of specifically prebound [125I]galanin in all tested regions. After such treatments, specific binding sites could be revealed in the hypothalamus namely in the parvocellular paraventricular nucleus, periventricular nucleus, arcuate nucleus and median eminence. In contrast, regions already labeled in standard conditions exhibited a slight decrease in [125I]galanin binding. Thus, regions were ranked from low to high rate of occupancy of galanin receptors by endogenous galanin, the rate of occupancy of galanin receptors being maximal in median eminence (greater than 90%). We thus studied the regional effect of guanine nucleotides on [125I]galanin specific binding. A high concentration (100 microM) of guanyl 5'-yl imidodiphosphate, a nonhydrolyzable analog of GTP directly added to the incubation medium, inhibited [125I]galanin binding in all telencephalic regions. On the same sections and only in regions of high index of galanin receptor occupancy (arcuate nucleus, median eminence, dorsomedial nucleus, paraventricular nucleus, and periventricular hypothalamic nucleus), guanyl 5'-yl imidodiphosphate paradoxically enhanced [125I]galanin binding. The effects of acid preincubation and guanyl 5'-yl imidodiphosphate incubation on [125I]galanin binding were strongly correlated in these hypothalamic areas (r = 0.97). In all regions, guanyl 5'-yl imidodiphosphate increased the rate of dissociation of [125I]galanin. In competition studies, guanyl 5'-yl imidodiphosphate decreased the IC50 s of unlabeled galanin which were homogenized around 4 nM in most telencephalic and hypothalamic regions. Thus, the guanyl 5'-yl imidodiphosphate-induced stimulation of [125I]galanin specific binding measured in the neuroendocrine and autonomic hypothalamus is linked to an increase in receptor capacity and not to a rise in receptor affinity. Both inhibitory and stimulatory guanyl 5'-yl imidodiphosphate effects observed in [125I]galanin equilibrium binding studies were dose-dependent and guanine nucleotide-specific with guanyl 5'-yl imidodiphosphate more potent than GTP or GDP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of rat olfactory bulb mitochondrial function by atrial natriuretic peptide

Summary Atrial Natriuretic Peptide (ANP) and receptors for ANP are widely distributed in many tissues and cell types in vertebrates. ANP has been shown to be internalized into the cytoplasm in several cell types and thus it raises the possibility that it may act on intracellular receptors. Displacement experiments of [¹²⁵I]-ANP binding to rat olfactory bulb mitochondrial fraction demonstrated the presence of high affinity (Kd<10^–9M) binding sites (Bmax, 112 fmol/mg protein) in this preparation. The addition of ANP (10^–8M) to this mitochondrial preparation resulted in a 25% increase in TPP⁺ accumulation, signifying a striking hyperpolarization of the mitochondrial membrane. In contrast ANP did not increase TPP⁺ uptake to liver mitochondrial preparations. This direct effect of ANP on Olfactory bulb mitochondrial membrane potential may underly the known effects of this hormone on steroidogenesis.     

Characterization and localization of ouabain receptors in Schistosoma mansoni

Saturable ouabain binding sites were detected in intact male schistosomes. The K_D for binding of ouabain to Schistosoma mansoni was 2.6 × 10^?7 M and to Schistosoma japonicum 2.9 × 10^?7 M. The binding of ouabain to the receptor demonstrated pharmacological specificity. Binding sites, obtained by differential centrifugation, were associated with fractions containing tegument (58%) and microsomal membranes (33%). Binding sites were concentrated in tegumental membranes, i.e., a 19-fold enrichment of receptors was found in membranes isolated from intact schistosomes exposed to Triton X-100. The antiparasitic drugs praziquantel (IC₅₀ = 9 × 10^?7 M) and Ro-11-3128 (IC₅₀ = 5 × 10^?6 M) inhibit binding of [³H]ouabain to intact parasites in a pharmacologic specific manner. Both praziquantel and Ro-11-3128 are without effect (IC₅₀ > 10^?4 M) on [³H]ouabain binding to homogenates of S. mansoni. These findings indicate that ouabain receptors are present in S. mansoni and that these receptors represent Na⁺-K⁺ pump sites. In addition, the characteristics and location of these receptors are consistent with previous observations on the physiological action of ouabain on the parasite.     

Distribution of nicotinic cholinergic receptors in the rat tel- and diencephalon: a quantitative receptor autoradiographical study using [3H]-acetylcholine, [α-125l]bungarotoxin and [3H]nicotine

The topographical distribution of [α:-¹²T]bungarotoxin [¹²⁵I]BTX, [³H]nicotine ([³H]Nic), [³H]acetylcholine ([³H]ACh) (in the presence of atropine) binding in rat teland diencephalon was investigated using a quantitative receptor autoradiographical technique. With the [³H|ACh and [³H]Nic radioligands, a strong labelling was observed in various thalamic nuclei, including the medial habenula, a moderate labelling in different areas of the cortex cerebri, the nucleus caudatus putamen, the nucleus accumbens and tuberculum olfactorium and a uniform weak labelling in the hypothalamus. When the binding data for [³H]Nic were plotted against binding data for [³H]ACh in various brain nuclei, a significant correlation was obtained. Considering [¹²⁵I]BTX, the strongest labelling was observed in the lateral mammillary nucleus and the hilus gyrus dentatus of the hippocampal formation. A weak labelling occurred in areas such as the nucleus causatus putamen, the thalamus and the cerebral cortex. No significant correlation was therefore obtained between the degree of [¹²⁵I]BTX binding in various brain nuclei and the degree of binding observed with [³H]Nic or [³H]ACh. The present results underline the view that the high-affinity |³H]Nic and [³H]ACh binding sites label the same cholinergic nicotinic receptor binding site, while [¹²⁵I]BTX labels another subpopulation of nicotinic cholinergic receptors, predominantly found in discrete areas of the hypothalamus and the limbic cortex.     

Atrial natriuretic peptide receptors along the rat and rabbit nephrons: [125I] α-rat atrial natriuretic peptide binding in microdissected glomeruli and tubules

Binding of [¹²⁵I] -rat atrial natriuretic peptide ([¹²⁵I] -RANP) was measured in glomeruli and pieces of tubule microdissected from rat and rabbit nephrons. High densities of specific ANP binding sites were found only in the glomeruli (10–30×10^–18 mol·glom^–1), whereas no specific binding could be detected in the proximal tubule, the thin segments of the Henle's loop, the thick ascending limb, the distal tubule and the cortical and outer medullary collecting tubules. Rising the temperature from 4° C to 35° C resulted in biphasic kinetics of binding, suggesting a temperature-dependent inactivation of labelled hormone by glomeruli. At 4° C, specific binding of [¹²⁵I] -RANP was time and dose-dependent and Scatchard analysis of data indicated an apparent equilibrium dissociation constant of 0.63 nM. Competition experiments revealed the following sequence of stereospecificity for binding to rat glomeruli: RANP 3–28>[¹²⁵I] -RANP=[¹²⁵I] -HANP=-RANP=atriopeptin III > atriopeptin II, whereas binding was unaffected by pharmacological doses of unrelated peptide hormones, prostaglandins, adrenergic agonists, dopamine, histamine and carbamylcholine. The results indicate that glomerular binding sites might be the physiological ANP receptors.Abbreviations ANP atria natriuretic peptide - 0RANP alpha rat atrial natriuretic peptide - PCT proximal convoluted tubule - PR pars recta - TDL thin descending limb - TAL thin ascending limb - MAL medullary thick ascending limb - CAL dortical thick ascending limb - DCT distal convoluted tubule - DCTb distal convoluted tubule bright - DCTg distal convoluted tubule granular - CCT conrtical collecting tubule - MCT outer medullary collecting tubule     

Evidence for a role of the neuropeptide galanin in spatial learning.

The neuropeptide galanin coexists with acetylcholine (ACh) in the basal forebrain cholinergic neurons and modulates cholinergic activity in the forebrain. The cholinergic forebrain neurons appear to play a significant role in learning and memory, as suggested by a severe loss of these neurons in Alzheimer's disease. The involvement of endogenous galanin in learning is demonstrated here by the use of the recently synthesized high-affinity galanin antagonist M35 [galanin(1-13)-bradykinin(2-9) amide] (Kd = 0.1 nM). Intracerebroventricular (i.c.v.) administration of M35 (6 but not 3 nmol) produced a significant (P < 0.025) facilitation of acquisition in a spatial learning test (Morris swim maze) without any increase in swim speed. Thus, M35 (6 nmol) shortened the escape latency, reduced the number of failures to reach the platform, and shortened the path length to reach the hidden platform. M35 (3 and 6 nmol) tended to enhance retention performance seven days after the last training session. Receptor autoradiographic studies on the distribution of [125I]M35 following i.c.v. administration show that it binds preferentially in the periventricular regions including the hippocampus. These results suggest that galanin may modulate spatial learning and memory and that galanin antagonists may provide a new principle in the treatment of Alzheimer's disease.     

Comparative distribution of binding of the muscarinic receptor ligands pirenzepine,AF-DX 384, (R,R)-I-QNB and (R,S)-I-QNB to human brain

Quinuclidinyl benzilate (QNB) and its derivatives are being developed to investigate muscarinic receptor changes in vivo in Alzheimer's disease and dementia with Lewy bodies. This is the first study of [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB binding in vitro in human brain. We have compared the in vitro binding of the muscarinic ligands [3H]pirenzepine and [3H]AF-DX 384, which have selectivity for the M1 and M2/M4 receptor subtypes, respectively, to the binding of [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB. This will provide a guide to the interpretation of in vivo SPET images generated with [123I]-(R,R)-I-QNB and [123I]-(R,S)-I-QNB. Binding was investigated in striatum, globus pallidus, thalamus and cerebellum, and cingulate, insula, temporal and occipital cortical areas, which show different proportions of muscarinic receptor subtypes, in post-mortem brain from normal individuals. M1 receptors are of high density in cortex and striatum and are relatively low in the thalamus and cerebellum, while M4 receptors are mainly expressed in the striatum, and M2 receptors are most evident in the cerebellum and thalamus. [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB density distribution patterns were consistent with binding to both M1 and M4 receptors, with [125I]-(R,R)-I-QNB additionally binding to a non-cholinergic site not displaceable by atropine. This distribution can be exploited by in vivo imaging, developing ligands for both SPET and PET, to reveal muscarinic receptor changes in Alzheimer's disease and dementia with Lewy bodies during the disease process and following cholinergic therapy.     

Binding of human EPF to receptors on PMBC as a first signal of pregnancy immunomodulation

The interaction between human early pregnancy factor (EPF) anda specific receptor present on human peripheral blood mononuclearcells (PMBC) has been assessed. EPF was radio-iodiated by thelactoperoxidase method to a high specific activity, and the[¹²⁵I]EPF obtained bound in a specific and saturable mannerto the receptors on PBMC. Saturation of the binding sites occurredat 10^–9 M. There were 6600 specific binding sites percell in cells partially purified from pregnant women and fewerin those from non-pregnant women. Unlabelled EPF was capableof competing for binding sites with [¹²⁵I]EPF, while the bindingof [¹²⁵I]EPF was not inhibited by human chorionic gonadotrophin.A new assay was used that permits the culture of PBMC and thedetection of the surface IgG expression in the same microplate.With this method the influence in vitro of human EPF on lymphocyteexpression was tested. The results demonstrated a specific inhibitionof surface IgG expression of PBMC using a very low concentrationof EPF (10 pg/ml).