Binding of DL-[3H]-alpha-Amino-3-Hydroxy-5-Methyl-Isoxazole-4-Propionic Acid (AMPA) to Rat Cortex Membranes Reveals Two Sites or Affinity States

Abstract

A method for measuring [³H]-AMPA binding in rat cortex membranes is described. Specific binding was saturable and accounted for 95% of total binding at 5 nM of [³H]-AMPA. Non linear curve fitting of [³H]-AMPA saturation isotherms suggested the presence of two binding sites: the high affinity site showed a pKd of 8.26 ± 0.07 (Kd = 5.49 nM) and a Bmax of 0.19 ± 0.03 pmol/mg protein, whereas the low affinity site indicated a pKd of 7.28 ± 0.05 (Kd = 52 nM) and a Bmax of 1.30 ± 0.23 pmol/mg protein. The pharmacological profile of [³H]-AMPA binding has been determined by studying a series of compounds in binding displacement experiments: Quisqualate was the most potent inhibitor of [³H]-AMPA binding (IC₅₀ = 9.7 nM), followed by AMPA (19 nM), CNQX, DNQX and L-Glutamate (272–373 nM). Kainate was a moderate displacer (6.2 μM); Ibotenic acid and glycine were very weak inhibitors (74 and 92 μM, respectively). CPP, GAMS and L-Aspartic acid showed IC₅₀-values of over 400 μM and MK-801, DL-AP5 and NMDA were almost inactive at the maximal concentration used in our experiments.     

Glucocorticoids inhibit precursor incorporation into protein in splenic lymphocytes by stimulating protein degradation and expanding intracellular amino acid pools

In the presence of tracer concentrations of extracellular leucine (5 μM), treatment of rat splenic lymphocyte suspensions in vitro with 1 μM dexamethasone for 2.5–4 h caused a 30–35% inhibition of [³H]leucine incorporation into protein. As the extracellular leucine concentration was raised to 5 mM, this inhibition was progressively reduced to 0–12%. This phenomenon correlated with a marked dependence on extracellular leucine concentration of the dexamethasone-dependent enlargement of free intracellular leucine pools in splenic lymphocytes: a 123% increase in pool size with tracer extracellular leucine; a 10% increase with 5 mM leucine. Varying extracellular leucine had no effect on: (1) nuclear [³H]dexamethasone binding by the cells; (2) the concentration of dexamethasone needed for half-maximal inhibition of [³H]leucine incorporation; (3) the time course of onset and maximal expression of the hormonal inhibition of [³H]leucine incorporation; or (4) the magnitude of dexamethasone-dependent inhibition of [³H]uridine incorporation into RNA by these cells. There was no detectable effect of dexamethasone on uptake and retention of [³H]leucine by the cells, regardless of the extracellular leucine concentration. Treatment of splenic lymphocytes for 4 h in vitro with 1 μM dexamethasone caused a small shift of ribosomes from larger aggregate polysomes to smaller forms. Thus, glucocorticoid-induced inhibition of amino acid incorporation in splenic lymphocytes is a multicomponent response, of which an actual decrease in protein synthesis is only a small part. Enlargement of free intracellular amino acid pools, probably resulting from increased protein degradation, is the major contributing factor to the hormonal inhibition of amino acid incorporation.     

Specific binding of an immunoreactive and biologically active 125I-labeled N(1)acylated substance P derivative to parotid cells

A biologically active ¹²⁵I-substance P derivative (I¹²⁵-BH-substance P), prepared by conjugation of substance P with [^125I]Bolton-Hunter reagent, binds specifically to isolated rat parotid cells. The Kd is 4 nM for I-BH-substance P, 5 nM for substance P, 0.18 μM for substance P octa(4–11)peptide, and 1.6 μM for substance P [pyroglutamyl⁶]hexa(6–11)peptide. Substance P free acid and substance P penta(7–11)peptide are much weaker competitors and the C-terminal tri(9–11)peptide has no effect at 30 μM. The binding is also inhibited by 1 μM physalaemin, eledoisin and substance P methyl ester, but not by unrelated peptides. The selective inhibition of the binding by the biologically active analogs and fragments of substance P indicates that the ¹²⁵I-labeled N(1)acylated substance P derivative may interact with a substance P receptor on parotid cells.     

Muscarinic receptor binding in rat brain using the agonist, [3H]cis methyldioxolane

Muscarinic receptor binding was measured in rat forebrain preparations using the muscarinic agonist, [³H]cis methyldioxolane ([³H]CD). The results of equilibrium binding studies using [³H]CD concentrations between 0.5–64 nM showed that [³H]CD binding did not saturate in this concentration range, although the binding isotherm was concave downward. Nonlinear regression analysis of the binding data revealed the presence of two populations of muscarinic receptors having dissociation constants of 1.83 and 123 nM and binding capacities of 85 and 1320 fmol/mg protein, respectively. Competitive inhibition experiments showed that [³H]CD binding was readily displaced by several muscarinic agonists and antagonists. The stereospecificity of [³H]CD binding was demonstrated in competitive inhibition experiments using the stereoisomers of benzetimide and acetyl-β-methylcholine. Dexetimide was 10,000 times more potent than levetimide and l-acetyl-β-methylcholine was 520 times more potent than d-acetyl-β-methylcholine. A variety of nonmuscarinic cholinergic drugs were not effective at inhibiting [³H]CD binding at a concentration of 10 μM.     

The fate of labelled glucose molecules in the rat adipocyte: Dependence on glucose concentration

Isolated rat adipocytes were incubated with 15 nM [3-³H]glucose or 100 nM [U-¹⁴C]glucose with or without insulin and in the absence or presence of unlabelled glucose. Following a 2 h incubation with 15 nM [3-³H]glucose, about two thirds of the cell-associated ³H-labelled metabolic products were hydrophilic largely anionic intermediates and about one third was lipids. The equivalent values were 40 and 60%, respectively, when using 100 nM [U-¹⁴C]glucose. The only ¹⁴C-labelled metabolite escaping to the incubation medium was ¹⁴CO₂, which accounted for about 15% of the rate of metabolism. Therefore, the rate of incorporation of 100 nM [U-¹⁴C]glucose into the cell-associated metabolites was quite a good measure of its net influx rate. The conversion of the two tracers to the sum of the metabolic products in cells treated with a maximally stimulating insulin concentration remained constant with glucose concentrations up to about 100 μM and then decreased progressively. The incorporation of radioactivity into the different metabolites varied markedly over the glucose concentration range 0–100 μM, presumably due to the saturation of different metabolic pools at different glucose concentrations. This variation was much less in cells not stimulated with insulin. Consequently, the maximal effect of insulin on the incorporation of the tracers into a given metabolite (e.g., labelled lipids) varied over the entire glucose concentration range. In addition, the apparent sensitivity (ED₅₀) with respect to the incorporation into a given metabolite was also dependent on the glucose concentration.     

Hormone action at the membrane level. IV. Epinephrine binding to rat liver plasma membranes and rat epididymal fat cells

[³H]Epinephrine binding to isolated purified rat liver plasma membrane is a reversible process. An initial peak in binding occurs at about 15 min and a plateau occurs by 50 min. Optimal binding occurred at a membrane protein concentration of 125μg. Rat liver plasma membranes stored at ?70 °C up to 4 weeks showed no difference in epinephrine binding capacity as compared to control fresh membranes.Epinephrine binding to liver plasma membranes was decreased by 79% by phospholipase A₂ (phosphatide acylhydrolase EC 3. 1. 1. 4), 81% by phospholipase C (phosphatidylcholine choline phosphohydrolase EC 3.1.4.3) and 59% by phopholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4). Trypsin and pronase digestion of the membrane decreased epinephrine binding by 97 and 47% respectively.In the presence of 10^?3M Mg²⁺ ions, increasing concentrations of GTP decreased epinephrine binding to liver plasma membranes. A maximal effect was demonstrated with 10^?5M GTP, representing an inhibition of 52% of the control. In a Mg²⁺-free system, epinephrine binding was unaffected by GTP. However, in a Mg²⁺-free system, increasing concentrations of ATP cause increasing inhibition of hormone binding. ATP at 10^-3 M reduced epinephrine binding to 28% of the control. GTP (10^?5M) was shown to inhibit epinephrine uptake rather than epinephrine release from the membrane.[³H]Epinephrine binding to isolated rat epididymal fat cells shows an initial peak within 5 min followed by a gradual rise which plateaus after 60 min. Epinephrine binding increased nearly linearly with increasing fat cell protein concentration (40–200 μg protein).GTP (10^?5M) and ATP (10^?4M) decreased epinephrine binding to rat epididymal fat cells by 41%. Nearly complete inhibition of binding was demonstrated with 10^?2?10^?3M ATP. Epinephrine analogs that contain two hydroxyl groups in the 3 and 4 position on the benzene ring act as inhibitors of [³H]epinephrine binding to rat adipocytes. Alteration of the epinephrine side chain has relatively little influence on binding. Analogs in which one of the ring hydroxyl groups is missing or methylated are poor inhibitors of [³H]epinephrine binding.Alpha-(phentolamine and phenoxybenzamine) and beta-(propranolol and dichorisoproterenol) adrenergic blocking agents were tested with respect to their ability to influence [³H]epinephrine binding and their influence on epinephrine-stimulated lipolysis. Only dichloroisoproterenol significantly inhibited epinephrine binding (by 25%). The two beta-adrenergic blocking agents caused an inhibition of epinephrine-stimulated glycerol release, with propranolol being most effective. Phentolamine and phenoxybenzamine had no significant effect on the epinephrine stimulation of glycerol release by fat cells.     

Estrogen modulation of angiotensin-stimulated phosphoinositide hydrolysis in slices of rat anterior pituitary

In this study, slices of rat anterior pituitary were prelabeled with [³H]myo-inositol and the ability of angiotensins II and III to stimulate [³H]phosphoinositide hydrolysis was characterized. When using tissue derived from ovariectomized rats, dose-response experiments revealed that angiotensin II significantly increases [³H]inositol monophosphate formation (in the presence of 10 mM LiCI) at concentrations of 10 nM and above. Maximal stimulation by angiotensin II was observed at 1 μM (228% of basal) and 50% maximal stimulation was at 10.8 ± 2.7 nM. Angiotensin III was less potent when compared to angiotensin II (maximal stimulation at 10 μM; 220% of basal: 50% maximal stimulation, 475 ± 159 nM). When using normal female rats, significant stimulation by angiotensin II was not observed until 1 μM angiotensin II. When ovariectomized rats were treated for 7 days with 17β-estradiol, increases in [³H]inositol monophosphate induced by 1 μM angiotensin II were significantly reduced when compared to sesame oil vehicle controls.This study shows that estrogen down-regulates angiotensin receptor coupling in the anterior pituitary. Moreover, it illustrates the influence of the hormonal state of the animal on the regulation of the effects of angiotensins in this tissue.     

Partial hepatectomy aggravates cyclosporin A-induced neurotoxicity by lowering the function of the blood-brain barrier in mice

AimsCyclosporin A, a calcineurin inhibitor, produces neurotoxicity with relatively high frequency in organ-transplanted patients. The aim of the present study was to clarify whether acute liver failure (ALF) simulated to the transient liver dysfunction at an early phase after liver transplantation increases the susceptibility to cyclosporin A-induced neurotoxicity through the blood–brain barrier (BBB) dysfunction.Main methodsThe right internal, left lateral and left internal lobes in male ddy mice were surgically excised under sodium pentobarbital anesthesia. Effect of cyclosporin A on harmine-induced tremors was examined and BBB permeability to ³[H]cyclosporin A was assessed in partially (70%) hepatectomized mice at postoperative days 1, 3 and 7.Key findingsPatrial hepatectomy aggravated harmine-induced tremors. Cyclosporin A (50 mg/kg, i.p.) markedly augmented harmine-induced tremors in partially hepatectomized mice at postoperative day 1. Consistent with these behavioral findings, the brain uptake of ³[H]cyclosporin A in mice injected with ³[H]cyclosporin A into the jugular vein at postoperative day 1 was significantly increased by partial hepatectomy compared with sham operation.SignificanceOur results indicate that ALF increases BBB permeability to cyclosporin A by lowering the function of P-glycoprotein and tight junctions, consequently leading to augmentation of cyclosporin A-induced neurotoxicity. The possibility that cyclosporin A increases the risk of neurotoxicity including tremors at an early phase of liver transplantation must be considered.     

Effects of the neoclerodane Hardwickiic acid on the presynaptic opioid receptors which modulate noradrenaline and dopamine release in mouse central nervous system

We have comparatively investigated the effects of Hardwickiic acid and Salvinorin A on the K⁺-evoked overflow of [³H]noradrenaline ([³H]NA) and [³H]dopamine ([³H]DA) from mouse hippocampal and striatal nerve terminals, respectively. The K⁺-evoked overflow of [³H]DA was inhibited in presence of Salvinorin A (100 nM) but not in presence of Hardwickiic acid (100 nM). Hardwickiic acid (100 nM) mimicked Salvinorin A (100 nM) in facilitating K⁺-evoked hippocampal [³H]NA overflow and the two compounds were almost equipotent. Facilitation of [³H]NA overflow caused by 100 nM Hardwickiic acid was prevented by the κ-opioid receptor (KOR) antagonist norbinaltorphimine (norBNI, 100 nM) and by the selective δ-opioid receptor (DOR) antagonist naltrindole (100 nM), but was not altered by 100 nM D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), a selective μ-opioid receptor (MOR) antagonist. We conclude that Hardwickiic acid modulates hippocampal [³H]NA overflow evoked by a mild depolarizing stimulus by acting at presynaptic opioid receptor subtypes.     

Modulation of GABA binding sites by CNS depressants and CNS convulsants

[³H]Muscimol binding at 23°C and muscimol stimulated [³H]flunitrazepam binding at 37°C to membranes of rat cerebral cortex have been investigated. In washed membrane preparations, 2 apparent populations of [³H]muscimol binding sites can be observed. At 23°C [³H]muscimol binding is more sensitive to inhibition by NaCl and by other salts than at 0°C. The CNS depressants etazolate and pentobarbital reversibly enhance [³H]muscimol binding and they increase the affinity of muscimol as a stimulator of [³H]flunitrazepam binding. Conversely the CNS convulsants picrotoxin, picrotoxinin and isopropylbicyclophosphate (IPTBO) reversibly interfere with [³H]muscimol binding when NaCl is present and these drugs antagonize the effects of etazolate. In the presence of NaCl, picrotoxin, picrotoxinin and IPTBO also decrease the apparent affinity of muscimol or GABA as stimulator of [³H]flunitrazepam binding. Binding of [³H]muscimol to GABA recognition sites of rat cerebral cortex is enhanced by Ag⁺, Hg⁺ and Cu²⁺ in μM concentrations, Ag⁺ being most potent. The effects of 100 μM AgNO₃ persist after repeated washing of the membranes. When membranes are pretreated with AgNO₃ only one apparent population of [³H]muscimol binding sites with high affinity (K_d: 6–8 nM) is found. In AgNO₃ pretreated membranes, the affinity of muscimol as stimulator of [³H]flunitrazepam binding is increased 18 times (EC₅₀ 14 nM) when compared to control membranes, (EC₅₀ 253 nM). In AgNO₃ pretreated membranes, etazolate, pentobarbital and IPTBO fail to perturb either [³H]muscimol binding or baseline and muscimol stimulated [³H]flunitrazepam binding. The results demonstrate that the apparent sensitivity of GABA binding sites of the GABA-benzodiazepine-picrotoxin receptor complex can be increased by etazolate and pentobarbital and decreased by picrotoxin and IPTBO. These drugs have in common that they interfere with [³H]dihydropicrotoxinin binding.     

Stimulation of EAAC1 in C6 glioma cells by store-operated calcium influx

This study investigated how modulation of intracellular calcium alters the functional activity of the EAAC1 glutamate transporter in C6 glioma cells. Pre-incubation of C6 glioma cells with the endoplasmic reticulum Ca²⁺ ATP pump inhibitor, thapsigargin (10 μM) produced a time-dependent increase in the V_max for d-[³H]aspartate transport that reached a maximum at 15 min (143% of control; P < 0.001) that was accompanied by increased plasma membrane expression of EAAC1 and was blocked by inhibition of protein kinase C. Pre-incubation of C6 glioma cells with phorbol myristate-3-acetate (100 nM for 20 min) also caused a significant increase in the V_max of sodium-dependent d-[³H]aspartate transport (190% of control; P < 0.01). In contrast, in the absence of extracellular calcium, thapsigargin caused a significant inhibition in d-[³H]aspartate transport that was not mediated by protein kinase C. Blockade of store-operated calcium channels with 2-aminoethoxydiphenyl borate (50 μM) or SKF 96365 (10 μM) caused a net inhibition of d-[³H]aspartate uptake. Co-incubation of C6 glioma cells with both thapsigargin and 2-aminoethoxydiphenyl borate (but not SKF 96365) prevented the increase in d-[³H]aspartate transport that was observed in the presence of thapsigargin alone. Furthermore, 2-aminoethoxydiphenyl borate, but not SKF 96365, reduced the increase in intracellular calcium that occurred following pre-incubation of the cells with thapsigargin. It is concluded that, in C6 glioma cells, stimulation of EAAC1-mediated glutamate transport by thapsigargin is dependent on entry of calcium via the NSCC-1 subtype of store operated calcium channel and is mediated by protein kinase C. In contrast, in the absence of store operated calcium entry, thapsigargin inhibits transport.     

3H-imipramine binding in membrane preparations from brains of insects (Periplaneta americana,Locusta migratoria) and mollusc (Helix pomatia)

1. The binding of [³H]-imipramine to brain membrane preparations of the insects, Periplaneta americana, Locusta migratoria and the mollusc, Helix pomatia was investigated.2. [³H]-Imipramine binding is similar in invertebrates and vertebrates although, in insects, the binding is independent of sodium.3. In the three invertebrate systems studied, [³H]-imipramine binding was inhibited by 5-hydroxytryptamine (5-HT) only at high concentrations (100 μM 5-HT caused 20–30% inhibition in insects and 10% inhibition in snail).4. Studies on binding and incorporation of [³H]-imipramine and [³H]-5-hydroxytryptamine indicate that the imipramine binding site differs from the 5-hydroxytryptamine recognition site of uptake.5. The [³H]-imipramine binding sites are not specific for 5-hydroxytryptamine and have similar affinity for dopamine.     

Presence of D2-dofamine Receptors in Human Term Placenta

Abstract

We studied the binding of [³H]-spiperone on human term placental membranes. This binding reached plateau level after 30 min incubation at 37°C and was reversed (t_1/2 ～ 5 min) by addition of an excess of unlabeled spiperone. Scatchard analysis of saturation experiments with increasing doses of [³H]-spiperone (0–25 nM) showed one class of high affinity binding sites with a dissociation constant (Kd) of 14 ± 2 nM and a maximal binding capacity (Bmax) of 222 ± 9 fmoles/mg protein. The affinity of 5 competitors was determined in competitive binding assays. The D₂-dopamine antagonists were the most potent inhibitors: Ki for spiperone and haloperidol were 8 ± 2 and 56 ± 22 nM respectively. Dopamine inhibited [³H]-spiperone binding with a Ki of 570 ± 50 μM whereas Schering 23390 (D₁ antagonist) and propranolol (β-adrenergic antagonist) were without effect. The binding was also inhibited by 100 μM GTPγS (38 ± 8% inhibition), indicating that the dopamine receptor is coupled with a GTP binding protein. These results demonstrate for the first time the presence of D₂-dopamine receptors in human placenta.     

Antimicrobial,cytotoxic, and insulin-releasing activities of the amphibian host-defense peptide ocellatin-3N and its L-lysine-substituted analogs

The host-defense peptide ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH₂), first isolated from the Caribbean frog Leptodactylus nesiotus, inhibited growth of clinically relevant Gram-positive and Gram-negative bacteria as well as a strain of the major emerging yeast pathogen Candida parapsilosis. Increasing cationicity while maintaining amphipathicity by the substitution Asp⁴→Lys increased potency against the microorganisms by between 4- and 16-fold (MIC ≤3 μM) compared with the naturally occurring peptide. The substitution Ala¹⁸→Lys and the double substitution Asp⁴→Lys and Ala¹⁸→Lys had less effects on potency. The [D4K] analog also showed 2.5- to 4-fold greater cytotoxic potency against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells (LC₅₀ values in the range of 12–20 μM) compared with ocellatin-3N but was less hemolytic to mouse erythrocytes. However, the peptide showed no selectivity for tumor-derived cells [LC₅₀ = 20 μM for human umbilical vein endothelial cells (HUVECs)]. Ocellatin-3N and [D4K]ocellatin-3N stimulated the release of insulin from BRIN-BD11 clonal β-cells at concentrations ≥1 nM, and [A18K]ocellatin-3N, at concentrations ≥0.1 nM. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 μM, indicating that plasma membrane integrity had been preserved. The three peptides produced an increase in intracellular [Ca²⁺] in BRIN-BD11 cells when incubated at a concentration of 1 μM. In view of its high insulinotropic potency and relatively low hemolytic activity, the [A18K] ocellatin analog may represent a template for the design of agents with therapeutic potential for the treatment of patients with type 2 diabetes.     

An evaluation of techniques for labelling the surface proteins of cultured mammalian cells

We have evaluated four techniques for labelling the surface proteins of cultured mammalian cells. The techniques are: (a) the lactoperoxide system; (b) the pyridoxal phosphate-[³H]borohydride system; (c) the [³H]4,4′-diisothiocyano-2,2′-dihydrostilbene disulfonate system and (d) the galactose oxidase-[³H]borohydride system. The subcellular distribution of radiolabel produced by these techniques has been evaluated by authoradiography at the light microscope level and by cellular fractionation. We find that while all four systems label the surface membranes in the majority of the cell population, they also heavily label internal sites in a small subpopulation of nonviable cells. The contribution of the internally labelled cells to further biochemical analysis may represent a severe problem in investigations which rely solely on surface labels for the study of plasma membrane organization     

Delta endotoxin inhibits a phosphatase in midgut epithelial membranes of Heliothis virescens

A vanadate- and delta endotoxin-sensitive phosphatase copurified with plasma membranes and brush border membranes from the midgut epithelium of Heliothis virescens. Phosphatase activity was stimulated under alkaline conditions. Total phosphatase activity was inhibited 60% in the midgut membranes by 360 nM delta endotoxin with a K_i = 76 nM. Brush border membranes were inhibited 75% by 1.47 μM delta endotoxin with a K_i = 64.7 nM. Vanadate (200 μM) completely inhibited the toxinsensitive alkaline phosphatase. A 72 kDa protein was phosphorylated when membranes were incubated with Mg²⁺ and [³²P]orthophosphate, and phosphorylation was inhibited by both vanadate and delta endotoxin, suggesting that destabilization of this phosphoprotein was responsible for phosphatase inhibition.     

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [³H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [³H]muscimol binding sites by excess(10^?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [³H]muscimol binding was potently inhibited by muscimol itself (IC₅₀, 2.5 nM), GABA (1C₅₀, 43 nM), isoguvacine (IC₅₀, 61 nM), and 3-aminopropanesulphonic acid (IC₅₀, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC₅₀, 800 nM). δ- Aminovaleric acid (IC₅₀, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC₅₀, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC₅₀, 23 μM) and p-proline (IC₅₀, 66 μM) also inhibited [³H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [³H]muscimol binding (IC₅₀≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [³H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.     

Comparative aspects of nitrobenzylthioinosine and dipyridamole inhibition of adenosine accumulation in rat and guinea pig synaptoneurosomes

Dipyridamole (DPR) and nitrobenzylthioinosine (NBI) inhibition of adenosine accumulation in synaptoneurosomes derived from rat cerebral cortex, rat cerebellum, guinea pig cerebral cortex and guinea pig cerebellum was investigated. The inhibition of adenosine accumulation by NBI was observed to be distinctly biphasic in both guinea pig and rat synaptoneurosomes. Such biphasic inhibition consisted of a nM potency component to inhibition, accounting for 20–30% of the maximum inhibition, and a μM potency component, accounting for the remaining 70–80% maximum inhibition. Such an inhibitory profile contrasts sharply with that of DPR which appears monophasic, with a mean IC₅₀ of between 10⁻⁷ M and 10⁻⁶ M, in all rat and guinea pig synaptoneurosomes preparations studied.Further differences between the potency of NBI and DPR in inhibiting [³H]adenosine accumulation were also noted. DPR was more potent in inhibiting [³H]adenosine accumulation in guinea pig cerebellar synaptoneurosomes than in cerebral cortex synaptoneurosomes. In rat synaptoneurosomes, the reverse selectivity was observed. DPR was also 2–6 fold (depending on brain region of comparison) more potent in inhibiting adenosine accumulation in guinea pig synaptoneurosomes than in inhibiting such accumulation in rat synaptoneurosomes. In contrast, NBI was approximately equipotent in inhibiting adenosine accumulation in rat and guinea pig synaptoneurosomes. Additional binding studies using [³H]NBI are also reported. The data presented are entirely consistent with the hypotheses that (1) NBI and DPR bind to functionally relevant sites and (2) there are different populations of nucleoside transporters in mammalian brain.     

Discrimination of Multiple [3H]5-Hydroxytryptamine Binding Sites by the Neuroleptic Spiperone in Rat Brain

Certain neuroleptic drugs, such as spiperone and (+) butaclamol, can discriminate between two populations of [³H]5-hydroxytryptamine ([³H]5-HT) binding sites in rat brain. The butyrophenone neuroleptic spiperone shows the greatest selectivity for these two binding sites, having at least a 3000-fold difference between its dissociation constants (2-12 nM versus 35,000 nM) for the high- and low-affinity sites, respectively. Inhibition of [³H]5-HT binding by spiperone in rat frontal cortex and corpus striatum yields distinctly biphasic inhibition curves with Hill slopes significantly less than unity. Results from nonlinear regression analysis of these inhibition studies were consistent with a two-site model in each brain region. In the frontal cortex the high-affinity neuroleptic sites comprised about 60% of the total [^3/H]5-HT binding sites whereas in the corpus striatum they accounted for only 20% of the sites. Furthermore, saturation studies of [³H]5-HT binding assayed in the absence or presence of 1 μM-spiperone (a concentration that completely blocks the high-affinity site while having minimal activity at the low-affinity site) reveal a parallel shift in the Scatchard plot with no change in the dissociation constant of [³H]5-HT, but a significant decrease (64% in frontal cortex or 28% in corpus striatum) in the number of specific binding sites. These observations are consistent with the existence of at least two populations of [³H]5-HT binding sites having a differential regional distribution in rat brain.     

Biosynthesis and transport of glycoproteins in the small intestinal epithelium of rats: I. Developmental change and effect of hypophysectomy

The biosynthesis and intracellular transport of glycoproteins in duodenal absorptive cells of intact rats at 6 and 24 days and hypophysectomized rats at 24 days of age were studied after 20 min intralumenal pulse-labeling of d-[³H]galactose, l-[³H]fucose, or d-[³H]mannose. Autoradiographic studies showed that the incorporation of sugars increased significantly in intact rats between 6 and 24 days. When rats were hypophysectomized at 6 days of age, the intestinal epithelium at 24 days incorporated d-[³H]galactose at a level significantly lower than that of intact rats at 24 days. Hypophysectomy also interfered with the developmental increase in d-[³H]mannose, but not in l-[³H]fucose, incorporation. Biochemical study indicated that the radioactivity in the lipid-free acid-precipitable glycoproteins in the intestine of 24-day-old intact rats at 20 min after d-[³H]galactose injection was 129% and 97% higher than that in 6-day-old rats and in 24-day-old hypophysectomized rats, respectively. The patterns of intracellular transport of newly synthesized galactosylated or fucosylated glycoproteins in all animal groups were similar; the labeled glycoproteins were initially present in the Golgi and were transported through the smooth endoplasmic reticulum to either the lateral membrane or the brush-border membrane within 60 min after the injection of labeled sugars. The proportion of labeled glycoproteins that migrated to the brush-border membrane, however, increased about twofold in the intact rats between 6 and 24 days of age at 60–240 min after d-[³H]galactose injection. Hypophysectomy interfered with developmental increase in the transport of glycoproteins from the apical cytoplasm to the brush-border membrane. It was concluded that the incorporation of monosaccharide precursors into glycoproteins and the porportion of newly synthesized galactosylated or fucosylated glycoproteins transported to the brush-border membrane increase during postnatal development. The developmental changes are regulated, at least partially, by the pituitary gland.