Solubilization of High-Affinity,Guanine Nucleotide-Sensitive μ-Opioid Receptors from Rat Brain Membranes

Abstract: High-affinity μ-opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane-associated receptors with morphine during solubilization in the detergent 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate appeared to stabilize the μ-opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound [³H][d -Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin ([³H]DAMGO), a μ-selective opioid agonist, with high affinity (K_D = 1.90 ± 0.93 nM; B_max = 629 ± 162 fmol/mg of protein). Of the membrane-associated [³H]-DAMGO binding sites, 29 ± 7% were recovered in the solubilized fraction. Specific [³H]DAMGO binding was completely abolished in the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate). The solubilized receptor also bound [³H]diprenorphine, a nonselective opioid antagonist, with high affinity (K_D = 1.4 ± 0.39 nM, B_max = 920 ± 154 fmol/mg of protein). Guanosine 5′-O-(3-thiotriphosphate) did not diminish [³H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 µM competed with [³H]diprenorphine for the solubilized binding sites; in contrast, [d -Pen²,d -Pen⁵]-enkephalin, a δ-selective opioid agonist, and U50488H, a κ-selective opioid agonist, failed to compete with [³H]diprenorphine for the solubilized binding sites at concentrations of <1 µM. In the absence of guanine nucleotides, the DAMGO displacement curve for [³H]diprenorphine binding sites better fit a two-site than a one-site model with K_Dhigh = 2.17 ± 1.5 nM, B_max = 648 ± 110 fmol/mg of protein and K_Dlow = 468 ± 63 nM, B_max = 253 ± 84 fmol/mg of protein. In the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate), the DAMGO displacement curve better fit a one- than a two-site model with K_D = 815 ± 33 nM, B_max = 965 ± 124 fmol/mg of protein.     

Activity of Ionotropic Glutamate Receptors in Retinal Cells: Effect of Ascorbate/Fe2+-Induced Oxidative Stress

Abstract: The effect of oxidative stress induced by the oxidant pair ascorbate/Fe²⁺ on the activity of ionotropic glutamate receptors was studied in cultured chick retina cells. The release of [³H]GABA and the increase of the intracellular free Na⁺ concentration ([Na⁺]_i), evoked by glutamate receptor agonists, were used as functional assays for the activity of the receptors. The results show that the maximal release of [³H]GABA evoked by kainate (KA; ～20% of the total) or AMPA (～11% of the total) was not different in control and peroxidized cells, whereas the EC₅₀ values determined for peroxidized cells (33.6 ± 1.7 and 8.0 ± 2.0 µM for KA and AMPA, respectively) were significantly lower than those determined under control conditions (54.1 ± 6.6 and 13.0 ± 2.2 µM for KA and AMPA, respectively). The maximal release of [³H]GABA evoked by NMDA under K⁺ depolarization was significantly higher in peroxidized cells (7.5 ± 0.5% of the total) as compared with control cells (4.0 ± 0.2% of the total), and the effect of oxidative stress was significantly reduced by a phospholipase A₂ inhibitor or by fatty acid-free bovine serum albumin. The change in the intracellular [Na⁺]_i evoked by saturating concentrations of NMDA under depolarizing conditions was significantly higher in peroxidized cells (8.9 ± 0.6 mM) than in control cells (5.9 ± 1.0 mM). KA, used at a subsaturating concentration (35 µM), evoked significantly greater increases of the [Na⁺]_i in peroxidized cells (11.8 ± 1.7 mM) than in control cells (7.1 ± 0.8 mM). A saturating concentration (150 µM) of this agonist triggered similar increases of the [Na⁺]_i in control and peroxidized cells. Accordingly, the maximal number of binding sites for (+)-5-[³H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate ([³H]MK-801) was increased after peroxidation, whereas the maximal number of binding sites for [³H]KA was not affected by oxidative stress. These data suggest that under oxidative stress the activity of the ionotropic glutamate receptors is increased, with the NMDA receptor being the most affected by peroxidation.     

Chronic Ethanol Increases the Cannabinoid Receptor Agonist Anandamide and Its Precursor N-Arachidonoylphosphatidylethanolamine in SK - N - SH Cells

Abstract : In an earlier study, we demonstrated that chronic ethanol (EtOH) exposure down-regulated the cannabinoid receptors (CB1) in mouse brain synaptic plasma membrane. In the present study, we investigated the effect of chronic EtOH on the formation of anandamide (AnNH), an endogenous cannabimimetic compound, and its precursor N-arachidonoylphosphatidylethanolamine (N-ArPE) in SK-N-SH cells that were prelabeled with [³H]arachidonic acid. The results indicate that exposure of SK-N-SH cells to EtOH (100 mM) for 72 h significantly increased levels of [³H]AnNH and [³H]N-ArPE (p < 0.05) (1.43-fold for [³H]AnNH and 1.65-fold for [³H]N-ArPE). Exposure of SK-N-SH cells to EtOH (100 mM, 24h) inhibited initially the formation of [³H]AnNH at 24 h, followed by a progressive increase, reaching a statistical significance level at 72 h (p < 0.05). [³H]N-ArPE increased gradually to a statistically significant level after 48 and 72 h (p < 0.05). Incubation with exogenous ethanolamine (7 mM) and EtOH (100 mM, 72 h) did not result in an additive increase in the formation of [³H]AnNH. The formation of [³H]AnNH and [³H]N-ArPE by EtOH was enhanced by the Ca²⁺ ionophore A23187 or by the depolarizing agent veratridine and the K⁺ channel blocker 4-aminopyridine. Further, the EtOH-induced formation of [³H]AnNH and [³H]N-ArPE was inhibited by exogenous AnNH, whereas only [³H]AnNH formation was inhibited by the CB1 receptor antagonist SR141716A and pertussis toxin, suggesting that the CB1 receptor and G_i/o protein mediated the regulation of AnNH levels. The observed increase in the levels of these lipids in SK-N-SH cells may be a mechanism for neuronal adaptation and may serve as a compensatory mechanism to counteract the continuous presence of EtOH. The present observation taken together with our previous results indicate the involvement of the endocannabinoid system in mediating some of the pharmacological actions of EtOH and may constitute part of a common brain pathway mediating reinforcement of drugs of abuse including EtOH.     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum

A study was made of the time course and kinetics of [³H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [³H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent K_m values for GABA neurons for high and low affinity uptake were 0.33 × 10⁻⁶ M and 41.8 × 10⁻⁴ M, respectively. For nonneuronal cells, the apparent K_m for high affinity uptake was 0.29 × 10⁻⁶ M. The apparent V_max values for GABA neurons for high and low affinity uptake were 28.7 × 10⁻⁶ mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent V_max for high affinity uptake was 0.06 × 10⁻⁶ mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 × 10⁻⁹ M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [³H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75–85%) of the [³H]GABA (4.4 × 10⁻⁶ M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.     

Characterization of an Adenylate Cyclase-Linked Serotonin (5-HT1) Receptor in a Neuroblastoma × Brain Explant Hybrid Cell Line (NCB-20)

Clonal cell line NCB-20 (a hybrid of mouse neuroblastoma N18TG2 and Chinese hamster 18-day embryonic brain expiant) expressed both high- (K_D 180 nM) and low-affinity (>3000 nM) binding sites for [³H]serotonin (5-HT) which were absent from the parent neuroblastoma. The low-affinity binding site was eliminated by 1 μM spiperone. The order of drug potency for inhibition of high-affinity [³H]5-HT binding was consistent with a 5-HT₁ receptor (5,6 - dihydroxytryptamine = 5-HT = methysergide = 5-methoxytryptamine > cyproheptadine = clozapine = mianserin > spiperone > dopamine = morphine = ketanserin = norepinephrine). [³H]5-HT binding was inhibited by guanine nucleotides (e.g., GTP and Gpp(NH)p), whereas antagonist binding was not; as-corbate was also inhibitory. A 30-min exposure of cells to 1—2 μM 5-HT or other agonists produced a three- to fivefold stimulation of cyclic AMP levels. The order of potency for 5-HT agonist stimulation of basal cyclic AMP levels and 5-HT antagonist reversal of agonist-stimulated levels was the same as the order of drug potency for inhibition of high-affinity [³H]5-HT binding, suggesting linkage of the 5-HT₁ receptor to adenylate cyclase in NCB-20 cells.     

Characterization and Distribution of a Cloned Rat μ-Opioid Receptor

Abstract: We have cloned and expressed a rat brain cDNA, TS11, that encodes a μ-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [³H]diprenorphine with high affinity (K_D = 0.23 ± 0.04 nM). The rank order potency of drugs competing with [³H]diprenorphine was as follows: levorphanol (K_i = 0.6 ± 0.2 nM) ≈β-endorphin (K_i = 0.7 ± 0.5 nM) ≈ morphine (K_i = 0.8 ± 0.5 nM) ≈ [d -Ala², N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAMGO; K_i = 1.6 ± 0.5 nM) ? U50,488 (K_i = 910 ± 0.78 nM) > [d -Pen^2,5]-enkephalin (K_i = 3,170 ± 98 nM) > dextrorphan (K_i = 4,100 ± 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar K_i are consistent with a μ-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) In COS-7 cells 50 µM 5′-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC₅₀ = 3.4 ± 0.5 nM) to a lower-affinity state (IC₅₀ = 89.0 ± 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned μ-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the μ-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of μ-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.     

Ionic Strength Dependence of Calcium, Adenine Nucleotide, Magnesium, and Caffeine Actions on Ryanodine Receptors in Rat Brain

Abstract: [³H]Ryanodine binding studies of ryanodine receptors in brain membrane preparations typically require the presence of high salt concentrations in assay incubations to yield optimal levels of binding. Here, radioligand binding measurements on rat cerebral cortical tissues were conducted under high (1.0 M KCI) and low (200 mM KCI) salt buffer conditions to determine the effects of ionic strength on receptor binding properties as well as on modulation of ligand binding by Ca²⁺, Mg²⁺, β,γ-methylene-adenosine 5′-triphosphate (AMP-PCP), and caffeine. In 1.0 M KCI buffer, labeled titration/equilibrium analyses yielded two classes of binding sites with apparent K_D (nM) and B_max (fmol/mg of protein) values of 2.4 and 34, respectively, for the high-affinity site and 19.9 and 157, respectively, for the low-affinity site. Unlabeled titration/equilibrium measurements gave a single high-affinity site with a K_D value of 1.9 nM and a B_max value of 95 fmol/mg of protein. The apparent K_D value derived from association and dissociation studies was 20 pM. Equilibrium binding was activated by Ca²⁺ (K_D/Ca²⁺= 14 nM), inhibited by Mg²⁺ (IC₆₀= 5.0 mM), and unaffected by AMP-PCP or caffeine. In 200 mM KCI buffer conditions, labeled titration analyses gave only a single site with a K_D value similar to and a B_max value 1.8-fold greater than those obtained for the low-affinity site in 1.0 M KCI buffer. In unlabeled titration measurements, the K_D value was fivefold lower, whereas the B_max value was unaffected. The K_D value derived from association and dissociation analysis was 2.4-fold greater in 200 mM KCI compared with 1.0 M KCI buffer conditions. In 200 mM compared with 1.0 M KCI, the potency with which Mg²⁺ inhibited binding was increased by 3.8-fold, whereas the affinity of the activation site for Ca²⁺ was reduced by 13-fold. Addition of caffeine in the presence of low salt increased the affinity of Ca²⁺ activation by 1.7-fold. The inhibitory effect of Mg²⁺ on [³H]-ryanodine binding in the presence of 200 mM KCI was reversed by AMP-PCP and caffeine with apparent EC₅₀ values of 0.25 and 7.6 mM, respectively. Taken together, these results indicate that ionic strength is an important consideration in binding studies of brain ryanodine receptors and their interactions with modulatory agents.     

Ionic Requirements for the Specific Binding of [3H]GBR 12783 to a Site Associated with the Dopamine Uptake Carrier

At 0°C, when Na⁺ was the only cation present in the incubation medium, increasing the Na⁺ concentration from 3 to 10 mM enhanced the affinity of [³H]l-[2-(di-phenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([³H]GBR 12783) for the specific binding site present in rat striatal membranes without affecting the 5_max. For higher Na⁺ concentrations, specific binding values plateaued and then slightly decreased at 130 mM Na⁺. In a 10 mM Na⁺ medium, the K_D and the B_max were, respectively, 0.23 nM and 12.9 pmol/mg of protein. In the presence of 0.4 nM [³H]GBR 12783, the half-maximal specific binding occurred at 5 mM Na⁺. A similar Na⁺ dependence was observed at 20°C. Scatchard plots indicated that K⁺, Ca²⁺, Mg²⁺, and Tris⁺ acted like competitive inhibitors of the specific binding of [³H]GBR 12783. The inhibitory potency of various cations (K⁺, Ca²⁺, Mg²⁺, Tris⁺, Li⁺ and choline) was enhanced when the Na⁺ concentration was decreased from 130 to 10 mM. In a 10 mM Na⁺ medium, the rank order of inhibitory potency was Ca²⁺ (0.13 mM) > Mg²⁺ > Tris⁺ > K⁺ (15 mM). The requirement for Na⁺ was rather specific, because none of the other cations acted as a substitute for Na⁺. No anionic requirement was found: Cl^-, Br^-, and F^- were equipotent. These results suggest that low Na⁺ concentrations are required for maximal binding; higher Na⁺ concentrations protect the specific binding site against the inhibitory effect of other cations.     

[3H]Bicuculline Methochloride Binding to Low-Affinity γ-Aminobutyric Acid Receptor Sites

Abstract: The binding of [³H]bicuculline methochloride (BMC) to mammalian brain membranes was characterized and compared with that of [³H]γ-aminobutyric acid ([³H]GABA). The radiolabeled GABA receptor antagonist showed significant displaceable binding in Tris-citrate buffer that was improved by high concentrations of chloride, iodide, or thiocyanate, reaching >50% displacement in the presence of 0.1 M SCN^?. An apparent single class of binding sites for [³H]BMC (K_D= 30 nM) was observed in 0.1 M SCN^? for fresh or frozen rat cortex or several regions of frozen and thawed bovine brain. The B_max was about 2 pmol bound/mg of crude mitochondrial plus microsomal membranes from unfrozen washed and osmotically shocked rat cortex, similar to that for [³H]GABA. Frozen membranes, however, showed decreased levels of [³H]BMC binding with no decrease or an actual increase in [³H]GABA binding sites. [³H]BMC binding was inhibited by GABA receptor specific ligands, but showed a higher affinity for antagonists and lower affinity for agonists than did [³H]GABA binding. Kinetics experiments with [³H]GABA binding revealed that low- and high-affinity sites showed a similar pharmacological specificity for a series of GABA receptor ligands, but that whereas all agonists had a higher affinity for slowly dissociating high-affinity [³H]GABA sites, bicuculline had a higher affinity for rapidly dissociating low-affinity [³H]GABA sites. This reverse potency between agonists and antagonists during assay of radioactive antagonists or agonists supports the existence of agonist- and antagonist-preferring conformational states or subpopulations of GABA receptors. The differential affinities, as well as opposite effects on agonist and antagonist binding by anions, membrane freezing, and other treatments, suggest that [³H]BMC may relatively selectively label low-affinity GABA receptor agonist sites. This study, using a new commercially available preparation of [³H]bicuculline methochloride, confirms the report of bicuculline methiodide binding by Mohler and Okada (1978), and suggests that this radioactive GABA antagonist will be a valuable probe in analyzing various aspects of GABA receptors.     

Messenger RNA Levels and Binding Sites of Muscarinic Acetylcholine Receptors in Gastrointestinal Muscle Layers from Healthy Dairy Cows

Acetylcholine interacts with muscarinic receptors (M) to mediate gastrointestinal (GI) smooth muscle contractions. We have compared mRNA levels and binding sites of M₁to M₅ in muscle tissues from fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows. The mRNA levels were measured by quantitative RT-PCR. The inhibition of [³H]-QNB (1-quinuclidinyl-[phenyl-4-³H]-benzilate) binding by M antagonists [atropine (M_{1 ? 5}), pirenzepine (M₁), methoctramine (M₂), 4-DAMP (M₃), and tropicamide (M₄)] was used to identify receptors at the functional level. Maximal binding (B_max) was determined through saturation binding with atropine as a competitor. The mRNA levels of M₁, M₂, M₃, and M₅ represented 0.2, 48, 50, and 1.8%, respectively, of the total M population, whereas mRNA of M₄ was undetectable. The mRNA levels of M₂ and of M₃ in the ileum were lower (P < 0.05) than in other GI locations, which were similar among each other. Atropine, pirenzepine, methoctramine, and 4-DAMP inhibited [³H]-QNB binding according to an either low- or high-affinity receptor pattern, whereas tropicamide had no effect on [³H]-QNB binding. The [³H]-QNB binding was dose-dependent and saturable. B_max in fundus, pylorus, and PLAC was lower (P < 0.05) than in the ELSC, and in the pylorus lower (P < 0.05) than in the ileum. B_max and mRNA levels were negatively correlated (r = -0.3; P < 0.05). In conclusion, densities of M are different among GI locations, suggesting variable importance of M for digestive functions along the GI tract.     

Characterization and Subcellular Localization of l-[3H]Carnitine Binding Sites in Rat Brain

Abstract: In the present study, we investigated the existence of a binding site for l -carnitine in the rat brain. In crude synaptic membranes, l -[³H]carnitine bound with relatively high affinity (K_D = 281 nM) and in a saturable manner to a finite number (apparent B_max value = 7.3 pmol/mg of protein) of binding sites. Binding was reversible and dependent on protein concentration, pH, ionic strength, and temperature. Kinetic studies revealed a K_off of 0.018 min^?1 and a K_on of 0.187 × 10^?3 min^?1 nM^?1. Binding was highest in spinal cord, followed by medulla oblongata-pons ≥ corpus striatum ≥ cerebellum = cerebral cortex = hippocampus = hypothalamus = olfactory bulb. l -[³H]Carnitine binding was stereoselective for the l -isomers of carnitine, propionylcarnitine, and acetylcarnitine. The most potent inhibitor of l -[³H]carnitine binding was l -carnitine followed by propionyl-l -carnitine. Acetyl-l -carnitine and isobutyryl-l -carnitine showed an affinity ～500-fold lower than that obtained for l -carnitine. The precursor γ-butyrobetaine had negligible activity at 0.1 mM. l -Carnitine binding to rat crude synaptic membrane preparation was not inhibited by neurotransmitters (GABA, glycine, glutamate, aspartate, acetylcholine, dopamine, norepinephrine, epinephrine, 5-hydroxytryptamine, histamine) at a final concentration of 0.1 mM. In addition, the binding of these neuroactive compounds to their receptors was not influenced by the presence of 0.1 mMl -carnitine. Finally, a subcellular fractionation study showed that synaptic vesicles contained the highest density of l -carnitine membrane binding sites whereas l -carnitine palmitoyltransferase activity was undetectable, thus excluding the possibility of the presence of an active site for carnitine palmitoyltransferase. This finding indicated that the localization of the l -[³H]carnitine binding site should be essentially presynaptic.     

Sequestration of Muscarinic Cholinergic Receptors in Permeabilized Neuroblastoma Cells

Abstract: The feasibility of using a permeabilized preparation of human SH-SY-5Y neuroblastoma cells for studies of muscarinic acetylcholine receptor (mAChR) sequestration has been evaluated. Exposure of cells permeabilized with digitonin, streptolysin-O, or the α-toxin from Staphylococcus aureus to oxotremorine-M (Oxo-M) for 30 min resulted in a 25–30% reduction in the number of cell surface mAChRs, as monitored by the loss of N[³H]methyl- scopolamine ([³H]NMS) binding sites. The corresponding value for intact cells was 40%. For cells permeabilized with 20 μM digitonin, the Oxo-M-mediated reduction in [³H]NMS binding was time (t_1/2～ 5 min) and concentration (EC₅₀～ 10 μM) dependent and was agonist specific (Oxo M > bethanechol = arecoline = pilocarpine). In contrast, no reduction in total mAChR number, as monitored by the binding of [³H]quinuclidinyl benzilate, occurred following Oxo-M treatment. The loss of [³H]NMS sites observed in the presence of Oxo-M was unaffected by omission of either ATP or Ca²⁺, both of which are required for stimulated phosphoinositide hydrolysis, but could be inhibited by the inclusion of guanosine 5′-O-(2-thiodiphosphate). mAChRs sequestered in response to Oxo-M addition were unmasked when the cells were permeabilized in the presence of higher concentrations of digitonin (80 μM). The results indicate (a) that permeabilized SH-SY-5Y cells support an agonist-induced sequestration of mAChRs, the magnitude of which is ～ 65–70% of that observed for intact cells, (b) that when internalized, mAChRs are located in a cellular compartment to which [³H]NMS has only a limited access despite the removal of the plasma membrane barrier, and (c) that the production of phosphoinositide-derived second messengers is not a prerequisite for mAChR sequestration.     

Regulation of hormone biosynthesis in cultured islet cells from anglerfish

Summary The effects of glucose and arginine on islet hormone biosynthesis were investigated using primary cell cultures prepared from islets of the anglerfish (Lophius americanus). After dispersion under sterile conditions, islet cells were maintained at 23° C in medium containing RPMI 1640 with Hanks' buffer, pH 7.5, modified by the adjustment of glucose (to 0.56 or 5.6 mM) and arginine (to 0.1, 1.15, or 10 mM) with the addition of 10% fetal bovine serum (dialyzed, heat inactivated) and penicillin/streptomycin. After 48 h, media were replaced by incorporation media containing [¹⁴C]isoleucine and [³H]tryptophan and incubated for an additional 8 h under otherwise identical conditions. Culture samples (cells plus media) were extracted, desalted, and gel filtered to identify and quantitate [¹⁴C]insulin, [³H]glucagon(s) plus [³H]somatostatin-28, and [³H]somatostatin-14 were In some experiments, [¹⁴C]insulin, [³H]glucagon(s), [³H]somatostatin-28, and [³H]somatostatin-14 were separated by high performance liquid chromatography. Raising the medium glucose from 0.56 (control) to 5.6 mM resulted in an augmentation in incorporation of [¹⁴C]isoleucine into insulin and an augmentation of [³H]tryptophan into glucagon(s) and somatostatin-14, but no change in incorporation of [³H]tryptophan into somatostatin-28. Raising the concentration of arginine from 0.1 to 1.15 or 10 mM resulted in a dose-dependent inhibition of labeled amino acid incorporation into all hormones except somatostatin-28. The results demonstrate the usefulness of the culture system for studying the modulation of hormone biosynthesis in anglerfish islet cells. This work was supported by Grants AM 16921 and AM 26378 from the National Institutes of Health, Bethesda, MD.     

[3H]Nitrobenzylthioinosine Binding as a Probe for the Study of Adenosine Uptake Sites in Brain

Abstract: The binding of the potent adenosine uptake inhibitor [³H]nitrobenzylthioinosine ([³H]NBI) to brain membrane fractions was investigated. Reversible, saturable, specific, high-affinity binding was demonstrated in both rat and human brain. The K_d in both was 0.15 nM with B_max values of 140–200 fmol/mg protein. Linear Scatchard plots were routinely obtained, indicating a homogeneous population of binding sites in brain. The highest density of binding sites was found in the caudate and hypothalamus in both species. The binding site was heat labile and trypsin sensitive. Binding was also decreased by incubation of the membranes in 0.05% Triton X-100 and by treatment with dithiothreitol and iodoacetamide. Of the numerous salt and metal ions tested, only copper and zinc had significant effects on [³H]NBI binding. The inhibitory potencies of copper and zinc were IC₅₀= 160 μM and 6 mM, respectively. Subcellular distribution studies revealed a high percentage of the [³H]NBI binding sites on synaptosomes, indicating that these sites were present in the synaptic region. A study of the tissue distribution of the [³H]NBI sites revealed very high densities of binding in erythrocyte, lung, and testis, with much lower binding densities in brain, kidney, liver, muscle, and heart. The binding affinity in the former group was approximately 1.5 nM, whereas that in the latter group was 0.15 nM, suggesting two types of binding sites. The pharmacologic profile of [³H]NBI binding was consistent with its function as the adenosine transport site, distinct from the adenosine receptor, since thiopurines were very potent inhibitors of binding whereas adenosine receptor ligands, such as cyclohexyladenosine and 2-chloroadenosine, were three to four orders of magnitude less potent. [³H]NBI binding in brain should provide a useful probe for the study of adenosine transport in the brain.     

Anesthetic Barbiturates Enhance Gsα-Dependent Cyclic AMP Production in S49 Mouse Lymphoma Cells

Abstract: Cyclic AMP (cAMP) regulates many important physiological processes. Barbiturates influence cAMP regulation, possibly through effects on G proteins. This study used intact S49 mouse lymphoma cells to characterize the role of G proteins in the effect of barbiturates on cAMP regulation. cAMP accumulation was determined in intact S49 WT (wild-type) and S49 cyc^? cells (the G_sα-deficient mutant) by measuring the conversion of [³H]-ATP to [³H]cAMP in cells preloaded with [³H]adenine. Pentobarbital enhanced cAMP accumulation in WT cells in the absence (basal) or presence of isoproterenol but had no effect on the EC₅₀ for isoproterenol. This effect was dose dependent with a 50–60% enhancement at 2 mM pentobarbital. Pentobarbital did not affect forskolin-stimulated cAMP accumulation in WT cells. In cyc^? cells, basal and forskolin-stimulated cAMP accumulation were stimulated only at the highest concentration of pentobarbital used (2 mM). Pentobarbital did not affect the inhibition of cAMP accumulation by somatostatin in WT cells, and pertussis toxin treatment of WT cells did not affect the action of pentobarbital on cAMP accumulation. Pentobarbital did not affect isoproterenol-stimulated adenylyl cyclase activity in whole-cell homogenates or membranes prepared from WT cells. The S-(?)-isomer of pentobarbital enhanced isoproterenol-stimulated cAMP accumulation more than the R-(+)-isomer. Phenobarbital and barbituric acid did not enhance isoproterenol-stimulated cAMP accumulation, whereas the anesthetic barbiturates hexobarbital, pentobarbital, and thiopental all enhanced activity. These results suggest that pentobarbital enhances cAMP accumulation in intact WT cells by a mechanism that is dependent on G_sα but independent of G_i. The properties of barbiturates that are responsible for the enhancement of cAMP accumulation may be related to the properties that are responsible for producing sedation and anesthesia.     

NMDA Receptors with Different Sensitivities to Magnesium and Ifenprodil Control the Release of [14C]Acetylcholine and [3H]Spermidine from Rat Striatal Slices in Vitro

Abstract: KCI (20–100 mM) and W-methyl-D-aspartate (NMDA, 100–1,000 μM) produce concomitant concentration-dependent increases in the release of previously captured [¹⁴C]acetylcholine and [³H]spermidine from rat striatal slices in vitro. The effects of NMDA (300μM) on striatal [¹⁴C]acetylcholine and [³H]spermidine release were blocked with equal potencies by the competitive NMDA antagonist CGP 37849, the glycine site antagonist L-689,560, and the NMDA channel blocker dizocilpine. In contrast, although NMDA-evoked [¹⁴C]acetylcholine release was antagonized by ifenprodil (IC₅₀= 5.3 μM) and MgCl₂, (IC₅₀= 200 μM), neither compound antagonized the NMDA-evoked release of [³H]spermidine at concentrations up to 100 μM (ifenprodil) or 1 mM (MgCl₂). Distinct NMDA receptor subtypes with different sensitivities to magnesium and ifenprodil therefore exist in the rat striaturn.     

Forskolin and 3-Isobutyl-l-Methylxanthine Increase Basal and Sodium Nitroprusside-Elevated Cyclic GMP Levels in Adult Guinea-Pig Cerebellar Slices

Abstract: In this study, the interaction between 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) in [³H]adenine-or [³H]-guanine-prelabelled adult guinea-pig cerebellar slices was investigated. Basal levels of [³H]cGMP were enhanced by forskolin, although no plateau was reached over the concentration range tested (0.1-100 μM). However, forskolin elicited a concentration-dependent, saturable potentiation of sodium nitroprusside (SNP)-stimulated [³H]cGMP accumulation (forskolin EC₅₀ value of 0.98 β 0.23 μM; 10 μM forskolin produced a 1.8 β 0.3-fold potentiation of the SNP response at 2.5 min). The forskolin potentiation was observed at all concentrations of SNP tested (0.001-10 mM). forskolin also elicited a large stimulation of [³H]-cAMP in [³H]adenine-prelabelled guinea-pig cerebellar slices; however, 1,9-dideoxyforskolin failed to elicit either a [³H]cAMP response or a potentiation of the SNP-induced [³H]cGMP response at concentrations up to 100 μM. Pretreatment with oxyhaemoglobin (50 μM) inhibited the response to SNP (1 mM) and forskolin (10 μM), as well as the response evoked by the combination of SNP and forskolih. A^G-Nitro-l -arginine (100 μM) inhibited the response to forskolin alone, but did not change the response to SNP or the potentiation induced by forskolin on SNP-induced [³H]cGMP levels. The protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 100 μM), staurosporine (10 μM), polymyxin B (100 μM), and Ro 31-8220 (10 μM) had no effect on the [³H]cGMP response to either SNP or the combination of SNP plus forskolin. N⁶,2′-Dibutyryl cAMP, at concentrations up to 10 mM, was also without effect on [³H]cGMP levels induced by SNP. 3-lso-butyl-1-methylxanthine reproduced the effect of forskolin on SNP-induced [³H]cGMP levels, but a less-than-additive effect was observed when the response to SNP was studied in the presence of forskolin and 3-isobutyl-1-methylxanthine. Taken together, these results infer that crosstalk between cyclic nucleotides takes place in guinea-pig cerebellar slices, and that cAMP may regulate cGMP-mediated responses in this tissue.     

Autoradiographic Distribution and Characteristics of High- and Low-Affinity Polyamine-Sensitive [3H]Ifenprodil Sites in the Rat Brain: Possible Relationship to NMDAR2B Receptors and Calmodulin

Abstract: We have studied the regional distribution and characteristics of polyamine-sensitive [³H]ifenprodil binding sites by quantitative autoradiography in the rat brain. In forebrain areas ifenprodil displaced [³H]ifenprodil (40 nM) in a biphasic manner with IC₅₀ values ranging from 42 to 352 nM and 401 to 974 µM. In hindbrain regions, including the cerebellum, ifenprodil displacement curves were monophasic with IC₅₀ values in the high micromolar range. Wiping studies using forebrain slices (containing both high- and low-affinity sites) or cerebellar slices (containing only the low-affinity site) showed that high- and low-affinity ifenprodil sites are sensitive to spermine and spermidine, to the aminoglycoside antibiotics neomycin, gentamicin, and kanamycin, and to zinc. Two calmodulin antagonists, W7 and calmidazolium, also displaced [³H]ifenprodil from both sites. Other calmodulin antagonists, including trifluoperazine, prenylamine, and chlorpromazine, selectively displaced [³H]ifenprodil from its low-affinity site in hindbrain and forebrain regions. High-affinity [³H]ifenprodil sites, defined either by ifenprodil displacement curves or by [³H]ifenprodil binding in the presence of 1 mM trifluoperazine, were concentrated in the cortex, hippocampus, striatum, and thalamus with little or no labeling of hindbrain or cerebellar regions. This distribution matches that of NMDAR2B mRNA, supporting data showing that ifenprodil has a preferential action at NMDA receptors containing this subunit. Low-affinity [³H]ifenprodil sites have a more ubiquitous distribution but are especially concentrated in the molecular layer of the cerebellum. [³H]Ifenprodil was found to bind to calmodulin-agarose with very low affinity (IC₅₀ of ifenprodil = 516 µM). This binding was displaced by calmodulin antagonists and by polyamines, with a potency that matched their displacement of [³H]ifenprodil from its low-affinity site in brain sections. However, the localization of the low-affinity [³H]ifenprodil site does not strictly correspond to that of calmodulin, and its identity remains to be further characterized. The restricted localization of high-affinity [³H]ifenprodil binding sites to regions rich in NMDAR2B subunit mRNA may explain the atypical nature of this NMDA antagonist.     

l-Glutamate Uptake Inhibitors May Stimulate Phosphoinositide Hydrolysis in Baby Hamster Kidney Cells Expressing mGluR1a via Heteroexchange with l-Glutamate Without Direct Activation of mGluR1a

Abstract: The functional efficacies of inhibitors of l -glutamate uptake for altering second messenger formation in baby hamster kidney cells expressing subtypes mGluR1a, mGluR2, and mGluR4 of the metabotropic glutamate receptor family were examined. l -Serine-O-sulfate was an agonist at mGluR1a (EC₅₀ = 70 µM), mGluR2 (EC₅₀ = 25 µM), and mGluR4 (EC₅₀ = 324 µM). l -Cysteine sulfinate, 1-aminocyclobutane-trans-1,3-dicarboxylate, l -cysteine, and dl -threo-3-methylaspartate stimulated phosphoinositide hydrolysis in mGluR1a cells with EC₅₀ values of 43, 64, 463, and 488 µM, respectively, and displaced l -[³H]glutamate binding from membranes prepared from these cells with respective IC₅₀ values of 48, 44, 79, and 139 µM. However, d -aspartate,l -trans-pyrrolidine-2,4-dicarboxylate, l -threo-3-hydroxyaspartate, and l -aspartate-β-hydroxamate stimulated phosphoinositide hydrolysis in mGluR1a cells (respective EC₅₀ values of 73, 54, 57, and 430 µM) but did not displace l -[³H]glutamate binding. These compounds inhibited Na⁺-dependent l -glutamate uptake into baby hamster kidney cells with IC₅₀ values similar to those for stimulation of phosphoinositide hydrolysis in mGluR1a cells. Phosphoinositide hydrolysis in mGluR1a cells, as stimulated by inhibitors of (or substrates for) this l -glutamate transporter, was significantly attenuated in the presence of l -glutamate decarboxylase (EC 4.1.1.15) or l -alanine aminotransferase (EC 2.6.1.2). Furthermore, incubation with 1 mMl -trans-pyrrolidine-2,4-dicarboxylate for 30 min increased the basal levels of free glutamate (1.5 ± 0.2 µM) in the assay buffer four- to fivefold as measured by HPLC analysis. Thus, heteroexchange with endogenous l -glutamate may lead to erroneous estimations of the functional efficacies at mGluR1a.     

Preservation of Redox, Polyamine, and Glycine Domains of the N-Methyl-D-Aspartate in Alzheimer's Disease

Abstract: This study used [³H] dizocilpine ([³H] MK-801) binding to the N-methyl-D-aspartate (NMDA) receptor to examine redox, polyamine, and glycine modulatory sites in membranes derived from the superior frontal and the superior temporal cortex of patients with Alzheimer's disease. In control subjects the competitive polyamine site antagonist arcaine inhibited [³H] dizocilpine binding in a dose-dependent fashion and this curve was shifted to the right by the addition of 50 μM spermidine. Arcaine inhibition of binding was more potent in the temporal cortex than in the frontal cortex, in both the absence and presence of 50 μMspermidine. In Alzheimer's disease, arcaine inhibition of [³H] dizocilpine binding (in both the absence and the presence of spermidine) was not different from control in either of the two brain areas examined. The sulfhydryl redox site of the NMDA receptor was assessed using the oxidizing agent 5, 5′-dithio-bis(2-nitrobenzoic acid), which inhibited binding in a dose-dependent fashion. This inhibition was similar in patients with Alzheimer's disease and control subjects. Glycine-stimulated [³H] dizocilpine binding was also unaffected in patients with Alzheimer's disease. However, in the temporal cortex there was a significant age-associated decline in [³H] dizocilpine binding in the presence of 100 μM glutamate (R₈=-0.71) and 100 μM glutamate plus 30 μM glycine (R₈=?0.90). There was also an age-related increase in arcaine IC₅₀ (which reflects an age-related decrease in arcaine affinity) in the frontal cortex, determined both in the absence (R₈= 0.83) and the presence (R₈= 0.79) of spermidine. These data indicate that the NMDA receptor and its modulatory redox, polyamine, and glycine subsites are intact in patients with Alzheimer's disease and that the modulatory activity of polyamine and glycine sites decline with aging.