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A single nucleotide polymorphism (SNP) in CHRNA5 (rs16969968, change from an aspartic acid [D] to asparagine [N] at position 398 of the human α5 nicotinic acetylcholine receptor subunit) has been associated with increased risk for nicotine dependence. Consequently, carriers of the risk variant may be at elevated risk for in utero nicotine exposure. To assess whether this gene‐environment interaction might impact nicotine intake in developmental nicotine‐exposed offspring, we utilized a mouse expressing this human SNP. D and N dams drank nicotine (100 μg/mL) in 0.2% saccharin water or 0.2% saccharin water alone (vehicle) as their sole source of fluid from 30 days prior to breeding until weaning of offspring. The nicotine (D Nic, N Nic) or vehicle (D Veh, N Veh) exposed offspring underwent a 2‐bottle choice test between postnatal ages of 30 to 46 days. N Nic offspring consumed the most nicotine at the highest concentration (400 μg/mL) compared with all other groups. In contrast, D Nic offspring drank the least amount of nicotine at all concentrations tested. Nicotine‐stimulated dopamine (DA) release measured from striatal synaptosomes was increased in D Nic offspring, while decreased in N Nic offspring relative to their genotype‐matched controls. These data suggest that the α5 variant influences the effect of developmental nicotine exposure on nicotine intake of exposed offspring. This gene‐environment interaction on striatal DA release may provide motivation for increased nicotine seeking in N Nic offspring and reduced consumption in D Nic offspring. [ABSTRACT FROM AUTHOR]

Firing rates of dopamine (DA) neurons in substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) control DA release in target structures such as striatum and prefrontal cortex. DA neuron firing in the soma and release probability at axon terminals are tightly regulated by cholinergic transmission and nicotinic acetylcholine receptors (nAChRs). To understand the role of α6* nAChRs in DA transmission, we studied several strains of mice expressing differing levels of mutant, hypersensitive (leucine 9' to serine [L9'S]) α6 subunits. α6 L9'S mice harboring six or more copies of the hypersensitive α6 gene exhibited spontaneous home-cage hyperactivity and novelty-induced locomotor activity, whereas mice with an equal number of WT and L9'S α6 genes had locomotor activity resembling that of control mice. α6-dependent, nicotine-stimulated locomotor activation was also more robust in high-copy α6 L9'S mice versus low-copy mice. In wheel-running experiments, results were also bi-modal; high-copy α6 L9'S animals exhibited blunted total wheel rotations during each day of a 9-day experiment, but low-copy α6 L9'S mice ran normally on the wheel. Reduced wheel running in hyperactive strains of α6 L9'S mice was attributable to a reduction in both overall running time and velocity. ACh and nicotine-stimulated DA release from striatal synaptosomes in α6 L9'S mice was well-correlated with behavioral phenotypes, supporting the hypothesis that augmented DA release mediates the altered behavior of α6 L9'S mice. This study highlights the precise control that the nicotinic cholinergic system exerts on DA transmission and provides further insights into the mechanisms and consequences of enhanced DA release., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Acetylcholine release stimulated by nicotinic agonists was measured as radioactivity released from perfused synaptosomes prepared from mouse interpeduncular nucleus (IPN) that had been loaded with [(3)H]choline. Agonist-stimulated release was dependent upon external calcium and over 90% of released radioactivity was acetylcholine. The release process was characterized by dose response curves for 13 agonists and inhibition curves for six antagonists. alpha-Conotoxin MII did not inhibit this release, while alpha-conotoxin AuIB inhibited 50% of agonist-stimulated release. Comparison of this process with [(3)H]dopamine release from mouse striatal synaptosomes indicated that different forms of nicotinic acetylcholine receptors (nAChRs) may mediate these processes. This was confirmed by assays using mice homozygous for the beta 2 subunit null mutation. The deletion of the beta 2 subunit had no effect on agonist-stimulated acetylcholine release, but abolished agonist-stimulated release of dopamine from striatal synaptosomes. Mice heterozygous for the beta 2 subunit null mutation showed decreased dopamine release evoked by L-nicotine with no apparent change in EC(50) value, as well as similar decreases in both transient and persistent phases of release with no changes in desensitization rates.

Nicotinic agonist-stimulated efflux of 86Rb+ from mouse brain synaptosomes was monitored continuously by on-line radioactivity detection. The concentration-effect curve following a 5-s stimulation with acetylcholine was biphasic (EC50 = 7.2 and 550 microM). alpha-Bungarotoxin (100 nM) did not inhibit the response, but dihydro-beta-erythroidine (DHbetaE) blocked both phases with differing potency (average IC50 =.22 and 8.9 microM for responses activated by low and high acetylcholine concentrations, respectively). Differential sensitivity DHbetaE inhibition was used to measure stimulation of 86Rb+ efflux by 17 nicotinic agonists, which differed markedly in potency and efficacy. All agonists were more potent at the DHbetaE-sensitive site. Both components were inhibited by the six antagonists tested. Methyllycaconitine and DHbetaE were more potent for the DHbetaE-sensitive component, whereas hexamethonium was more potent at the DHbetaE-resistant component. Both DHbetaE-sensitive and DHbetaE-resistant responses were reduced more than 95% in beta2-null mutant mice, establishing the requirement for the beta2 subunit for both components. Both components were widely, but not identically, distributed throughout the brain. The DHbetaE-sensitive component appears to be identical with agonist-stimulated 86Rb+ efflux described previously and is likely to be mediated by alpha4beta2 receptors. The DHbetaE-resistant component is a novel, active, and widely distributed response mediated by nicotinic receptor(s) that also require the beta2 subunit.

L-Nicotine stimulates a biphasic release of [3H]dopamine from mouse striatal synaptosomes which does not persist after agonist is removed. Approximately 80% of the initial release is transient and disappears with a half-time of less than 1 min; the other 20% persists for several minutes (t(1/2), 5-10 min). Both the transient and persistent phases were investigated by 10-min exposures to agonists with an in vitro perfusion technique. A series of nicotinic agonists and antagonists were used to determine the pharmacological relationship of the two phases. Parameters measured included EC50 and Vmax values and desensitization rates for both phases for agonists, Ki values for antagonists and Ki values for low concentrations of agonists. The results are consistent with both phases being mediated by a single type of receptor. In addition, the effects of chronic nicotine treatment on transient and persistent [3H]DA release were measured. For both phases, release was decreased approximately 15% by chronic infusion of 4.0 mg/kg/hr L-nicotine. Correlation of the results with inactivation of a portion of the receptors rather than a reversible desensitization is discussed.

Progesterone and its A-ring reduced metabolites are allosteric activators of GABA(A) receptors. The studies reported here examined the effects of these steroids on brain nicotinic receptors using an 86Rb+ efflux assay that likely measures the function of alpha4beta2-type nicotinic receptors and [3H]dopamine release, which may be modulated by an alpha3-containing nicotinic receptor. Both of the A-ring reduced metabolites of progesterone were noncompetitive inhibitors of both assays, whereas progesterone inhibited only the 86Rb+ efflux assay. The 86Rb+ efflux assay was slightly more sensitive than was the dopamine release assay to steroid inhibition. Inhibition developed slowly for both assays (t1/2 = 0.4 min) and was reversed even more slowly (t1/2 = 10-15 min). Steroid addition did not alter either the rate of association of [3H]nicotine binding to brain membranes, nor was equilibrium binding changed. These findings argue that neurosteroids are allosteric inhibitors of brain nicotinic receptors.

The desensitization of nicotine-stimulated 86Rb+ efflux from synaptosomes prepared from C57BL/6 mouse brain was investigated. Nicotine stimulated a saturable, concentration-dependent efflux of 86Rb+ from synaptosomes (EC50 = 0.60 microM), but the response decreased with time of exposure to nicotine. The rate of decrease of the response (desensitization) increased as the nicotine concentration was increased (EC50 = 0.35 microM; maximal rate of desensitization = 1.1 min-1). Desensitization of nicotine-stimulated 86Rb+ efflux was also observed when synaptosomes were exposed to low (1-200 nM) concentrations of nicotine that caused little or no stimulation of efflux (EC50 = 13 nM). The rate of desensitization observed with low nicotine concentrations (0.30 min-1) was less than that measured at stimulating concentrations. Desensitization was not fully reversible for synaptosomes exposed to nicotine concentrations between 10 nM and 10 microM: Only 60-40% of the control response was regained after a 10-min washout period. The kinetics of functional desensitization were compared with the kinetics of [3H]nicotine binding. [3H]Nicotine binding to midbrain particulate fractions displayed both a fast and a slow phase. The EC50 values for these two phases were 2.6 and 14 nM, respectively. Data obtained from functional desensitization and ligand binding experiments were analyzed using a two-state model. The kinetic constants obtained from the analyses of these two processes were very similar. Overall, the results suggest that nicotinic receptor function measured with ion flux desensitizes when exposed to either stimulating or nonstimulating concentrations of nicotine. In addition, the kinetic properties calculated for the functional desensitization are comparable to those for [3H]nicotine binding.

Potential desensitization of brain nicotinic receptors was studied using a [3H]dopamine release assay. Nicotine-stimulated [3H]dopamine release from mouse striatal synaptosomes was concentration-dependent with an EC50 of 0.33 +/- 0.13 microM and a Hill coefficient of 1.44 +/- 0.18. Desensitization by activating concentrations of nicotine had a similar EC50 and a half-time of 35 s. Concentrations of nicotine that evoked little release also induced a concentration-dependent desensitization (EC50 = 6.9 +/- 3.6 nM, t1/2 = 1.6-2.0 min, nH = 1.02 +/- 0.01). Both types of desensitization produced a maximum 75% decrease in [3H]dopamine release. Recovery from desensitization after exposure to low or activating concentrations of nicotine was time-dependent with half-times of 6.1 min and 12.4 min, respectively. Constants determined for binding of [3H]nicotine to striatal membrane at 22 degrees C included a KD of 3.7 +/- 0.5 nM, Bmax of 67.5 +/- 2.2 fmol/mg, and Hill coefficient of 1.07 +/- 0.06. Association of nicotine with membrane binding sites was biphasic with half-times of 9 s and 1.8 min. The fast rate process contributed 37% of the total reaction. Dissociation was a uniphasic process with a half-time of 1.6 min. Comparison of constants determined by the release and binding assays indicated that the [3H]-nicotine binding site could be the presynaptic receptor involved in [3H]dopamine release in mouse striatal synaptosomes.

Two sensitive assays, one which fluorometrically measures only the L isomer of 2-keto-4-hydroxyglutarate after decarboxylation to L-malate and the other which spectrophotometrically determines both enantiomers by reductive amination with glutamate dehydrogenase, are described. By use of these assays, the steady-state kinetics of the aldol condensation of pyruvate with glyoxylate, as catalyzed by 2-keto-4-hydroxyglutarate aldolase from either bovine liver or Escherichia coli, were studied as was the inhibition of this reaction by glyoxylate and other anions. For the E. coli aldolase, double-reciprocal plots are linear except at high (above 5 mM) glyoxylate concentrations; apparent Km values increase with increasing concentrations of the fixed substrate. The data are consistent with an ordered reaction sequence. Inhibition by halides follows the lyotropic or Hofmeister series. Esters are not good inhibitors; mono-, di-, and tricarboxylic acids are increasingly inhibitory. Of the substrate analogues tested, hydroxypyruvate is the most potent inhibitor. Inhibition studies with citrate, acetaldehyde, and glyoxylate (all competitive inhibitors) suggest there are two domains at the active site-the Schiff base forming lysyl residue which interacts with carbonyl analogues (like acetaldehyde) and a center of positive charge which binds anions (like citrate). In contrast to the bacterial enzyme, liver 2-keto-4-hydroxyglutarate aldolase is inhibited in a competitive manner by much lower concentrations (0.1 mM or even lower) of glyoxylate. Many salts and some carboxylic acids activate the liver enzyme. Similarly, substrate analogues like 2-ketobutyrate and fluoropyruvate are mild activators; no effect is seen with acetaldehyde. Besides glyoxylate, only glyoxal, 2-ketoglutarate, and hydroxypyruvate inhibit the aldol condensation reaction. A uniform value of 1 is found for the number of inhibitor molecules bound per active site of either liver or E. coli 2-keto-4-hydroxyglutarate aldolase.

Metastatic variant sublines of the murine RAW117 large cell lymphoma or lymphosarcoma have been established in vitro by sequential cycles of harvesting of liver tumor nodules after intravenous inoculation of tumor cell suspensions into syngeneic BALB/c mice. After five and ten in vivo selections for liver colonization, variant sublines RAW117-H5 and -H10, respectively, were established, and these formed significantly more surface liver tumors than the parental RAW117-P line. RAW117 sublines were tested for their abilities to adhere to embryonic mouse liver or brain cells in an in vitro cell-cell adhesion assay. Liver colonizing RAW117-H10 cells adhered with greater selectivity to liver cells than to brain cells. Parental RAW117-P cells were more homotypically adhesive, but they were nonselective in their organ cell adhesion properties. We examined RAW117 cells for the presence of liver cross-reactive antigens using polyclonal xenoantibody preparations directed against embryonic murine liver cells. These antibody preparations block organ-specific homotypic adhesion of embryonic murine liver cells in vitro. The amount of fetal liver antigen(s) expressed on RAW117 sublines correlated with liver colonization potentials (H10 greater than H5 greater than P) in quantitative absorption assays. Treatment of the highly metastatic RAW117-H10 subline with polyclonal anti-embryonic murine liver F(ab')2 or Fab' antibody fragments had no effect on RAW117-H10 cell viability or growth in vitro or in vivo, but inhibited liver colonization (median liver tumor colonies reduced from greater than 200 to 0) and prolonged life expectancy. In contrast, pretreatment of RAW117-H10 cells with polyclonal anti-H-2 did not modify the in vivo biologic properties of these metastatic cells.

Rabbit antisera prepared against embryonic chick liver cells have been characterized using cell cytotoxicity tests, antibody absorption tests, inhibition of cell-cell reaggregation and cell-aggregate adhesion assays. The sera contain antibodies which specifically block embryonic chick liver cell-cell aggregation with no effect on other cell types or embryonic liver cells of other classes tested. Assays with sera absorbed with live cells, neural retina cells, or erythrocytes as well as with sera raised against these cells, indicate that this anti-aggregation effect is not merely the result of coating the cell surface with non-specific antibody. The anti-aggregation specificities of the sera have been checked by absorption experiments with a variety of membrane preparations. These results suggest significant cross-reactivity only between chick liver and kidney, perhaps reflecting the relatedness of the epithelial elements of these tissues. The immunoglobulin G fraction, F(ab')2 and monomeric fragments Fab' retain activity suggesting that the antisera inhibit cell-cell adhesion via blockage of cell surface ligands and not by cell surface modulation effects or cytotoxicity. The organ and class specificities of the antisera suggest that the inhibition of cell-cell aggregation is mediated by the direct binding of antibody to the embryonic chick liver cell adhesion ligand system.

Tritiated water and tritiated substrates have been used to study exchange reactions catalyzed by Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16, 2-oxo-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate). With pyruvate, the enzyme catalyzes a rapid first-order exchange of all three methyl hydrogens in the absence of added acceptor aldehyde (i.e. glyoxylate). This reaction is not rate limiting for aldol condensation or cleavage; quite different pH-activity profiles for the exchange reaction versus aldol cleavage and also comparative effects that pH changes have on Km and V values for the two processes favor this conclusion. The exchange reaction with 2-oxobutyrate, a substrate analog, is stereoselective; one methylene hydrogen is removed at a 6-fold faster rate than the other but eventually both are exchanged. No tritium exchange occurs with glyoxylate.