Studies of persistent infection by Chlamydia trachomatis serovar K in TPA-differentiated U937 cells and the role of IFN-gamma

To investigate the effects of adenosine A1 receptor activation on energy metabolism and RNA and protein biosynthesis in central neurons, cultured neurons from the rat forebrain were exposed for 1 hr to 72 hr to various concentrations (10 nM-100 microM) of the selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) or the A1 receptor antagonist 8-cyclopentyltheophylline (CPT). At all concentrations tested, the adenosinergic compounds did not affect cell viability within 72 hr of treatment, except for CPT, which reduced viability by 19.7% when used at the concentration of 100 microM. Energy metabolism was analysed by studying the specific uptake of 2-D-[3H]deoxyglucose ([3H]2DG). Rates of RNA and protein biosynthesis were assessed by the measurement of [3H]uridine and [3H]leucine incorporation, respectively. Neuronal [3H]2DG uptake was increased by 16% (P < 0.01) after 8 hr in the presence of 100 microM CCPA, whereas 100 microM CPT for 24 hr also increased [3H]2DG uptake (8%, P < 0.01). At these concentrations, both ligands inhibited [3H]uridine incorporation after a 3-hr treatment by 92% and 30%, respectively. CCPA never altered [3H]leucine incorporation when compared to controls, and CPT significantly inhibited protein synthesis only at 10-100 microM. Additional experiments to analyse the influence of A1 ligands on the transport of [3H]2DG, [3H]leucine and [3H]uridine suggested that CCPA and CPT, which interact functionally with adenosine receptors by regulating cyclic AMP production in this model, are able to alter energy metabolism and RNA synthesis in central neurons in a nonspecific manner by interacting with glucose and uridine transporters.     

Nitric oxide involvement in regulating the dopamine transport in the striatal region of rat brain

Spontaneous [3H]dopamine ([3H]DA) overflow was measured from striatal slices in the presence of different glutamate (Glu) receptor agonists such as N-methyl-D-aspartate (NMDA), kainate (KA) and quisqualate (QA) and their corresponding antagonists, Dizocilpine maleate (MK-801), D-gamma-glutamyl-aminomethanesulfonic acid (GAMS) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. [3H]DA uptake and release in the presence of L-Arginine (L-Arg) and NG-nitro-arginine (L-N-Arg), an inhibitor of nitric oxide (NO) synthesis were also evaluated. L-N-Arg alone or combined with L-Arg significantly reduced [3H]DA uptake at 10 and 100 microM from 33% to 44% from striatal slices. Whereas, in brain synaptosomal fractions L-Arg induced a biphasic effect on that [3H]DA uptake in a dose dependent manner, and L-N-Arg showed an absolute inhibition in 80-90% of this [3H]DA uptake at 1-500 microM. The amino acids, lysine, valine and histidine (100 microM) had a little effect inhibitory on [3H]DA uptake from synaptosomal fractions. Glu agonists, NMDA (10 microM) and KA (10 microM) importantly increased the spontaneous [3H]DA overflow, which was blocked by MK-801 (10 microM) and GAMS (10 microM), respectively. QA had no effect on [3H]DA release. L-Arg (10-200 microM) potentiated the spontaneous [3H]DA overflow in a dose dependent fashion from striatal slices, being reverted by 10 microM L-N-Arg alone or in combination with all other compounds; whereas, lysine, histidine and valine did not modify that spontaneous [3H]DA overflow. Results support the hypothesis related to the participation of NO on DA transport possibly synthesized at the dopaminergic (DAergic) terminals in the striatum; also that L-Arg concentration may determine alternative mechanisms to regulate the DAergic activity at the striatum.     

Transient immunosuppression allows transgene expression following readministration of adeno-associated viral vectors

The effects of external pH (pHout) variations on the Na+ and on the Ca2+ dependent fractions of the evoked amino acid neurotransmitter release were separately investigated, using GABA as a model transmitter. In [3H]GABA loaded mouse brain synaptosomes, the external acidification (pHout 6.0) markedly decreased the Na+ dependent fraction of [3H]GABA release evoked by veratridine (10 microM) in the absence of external Ca2+, as well as the Ca2+ dependent fraction of [3H]GABA release evoked by high (20 mM) K+ in the absence of external Na+. The depolarization-induced elevation of [Na(i)] (monitored in synaptosomes loaded with the Na+ indicator dye, SBFI) and the depolarization-induced elevation of [Ca(i)] (monitored in synaptosomes loaded with the Ca2+ indicator dye fura-2) were also markedly decreased at pHout 6. On the contrary, the external alkalinization (pHout 8) facilitated all the above responses. A slight increase of the baseline release of the [3H]GABA was observed when pHout was changed from 7.4 to 8. This effect was only observed in the presence of Ca2+. pHout changes from 7.4 to 6 or to 7 did not modify the baseline release of the transmitter. All the effects of pHout variations on [3H]GABA release were independent on the presence of HCO3-. It is concluded that external H+ regulate amino acid neurotransmitter release by their actions on presynaptic Na+ channels, as well as on presynaptic Ca2+ channels.     

Demonstration of glutamate/aspartate uptake activity in nerve endings by use of antibodies recognizing exogenous D-aspartate

Nerve terminals as well as glial cells are thought to possess high-affinity Na(+)-dependent transport sites for excitatory amino acids. However, recent immunocytochemical results with antibodies against such a transporter isolated from rat brain showed a selective labelling of glial cells [Danbolt et al. (1992) Neuroscience 51, 295-310]. Critical evaluation of the literature indicates that previous evidence for nerve terminal uptake of acidic amino acids might possibly be attributed to glia. To find out whether there is indeed a glutamate transporter in nerve endings, we incubated hippocampal slices with D-aspartate (10 and 50 microM), a metabolically inert substrate for the high-affinity glutamate transport system. After fixation by glutaraldehyde/formaldehyde the slices were processed immunocytochemically with specific polyclonal antibodies raised against D-aspartate coupled to albumin by glutaraldehyde/formaldehyde. The electron-microscopic postembedding immunogold technique demonstrated a large accumulation of gold particles in nerve terminals making asymmetrical synapses, compared to their postsynaptic dendritic spines, as well as in glial cell processes. The labelled terminals include those of the glutamatergic Schaffer collaterals. Axosomatic boutons appeared unlabelled. Comparison with a test conjugate with known concentration of fixed D-aspartate (94 mM) suggests that the concentration attained in the terminals after incubation with 50 microM D-aspartate was in the lower millimolar range. The uptake was totally dependent on Na+, blocked by L-threo-3-hydroxyaspartate, and had a high affinity for D-aspartate (apparent Km about 20 microM). There was no labelling in slices incubated without D-aspartate. Compared to glia, the nerve terminals had a higher D-aspartate density and accounted for a much higher proportion of the total tissue uptake, but this relationship may be different in vivo. At the light-microscopic level the D-aspartate-like immunoreactivity showed a distinct laminar distribution, identical to that shown autoradiographically for D-[3H]aspartate and L-[3H]glutamate uptake sites [Taxt and Storm-Mathisen (1984) Neuroscience 11, 79-100], and corresponding to the terminal fields of the major excitatory fibre systems in the hippocampal formation. The novel approach described here establishes that glutamatergic nerve terminals as well as glia do sustain sodium-dependent high-affinity transport of excitatory amino acids, implying that more than one glutamate transporter must be present in the brain. Immunogold detection of D-aspartate gives a much higher anatomical resolution than electron microscopic autoradiography of D-[3H]aspartate or L-[3H]glutamate uptake, the only method that has been available previously for ultrastructural demonstration of uptake activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heterologous expression of rat epitope-tagged histamine H2 receptors in insect Sf9 cells

1. Rat histamine H2 receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H2 receptor were prepared and were used to infect insect Sf9 cells. 2. The His-tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]-aminopotentidine (APT) binding studies. 3. In Sf9 cells expressing the His-tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1+/-0.1 microM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60+/-0.43 microM, 0.25+/-0.15 microM and 28+/-7 nM, respectively (mean+/-s.e.mean, n=3). 4. The expression of the His-tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6+/-0.6 pmol mg(-1) protein (mean+/-s.e.mean, n=3) after 3 days of infection. This represents about 2 x 10(6) copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-beta-cyclodextrin complex resulted in an increase of [125I]-APT binding up to 169+/-5% (mean+/-s.e.mean, n=3). 5. The addition of 0.03 mM cholesterol-beta-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC50 value of histamine was 3.1+/-1.7 microM in the absence of cholesterol-beta-cyclodextrin complex and 11.1+/-5.5 microM in the presence of cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 microM histamine was identical, 85+/-18 pmol/10(6) cells in the absence and 81+/-11 pmol/10(6) cells in the presence of 0.03 mM cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). 6. Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well. 7. These experiments demonstrate the successful expression of His-tagged histamine H2 receptors in insect Sf9 cells. The H2 receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.     

Upregulation of Na(+)-dependent alanine transport in vascular endothelial cells by serum: role of intracellular Ca2+

OBJECTIVE: Amino acid transport and its regulation in vascular endothelial cells remains a largely unexplored area. In this study, we evaluated alanine transport in bovine aortic endothelial cells to assess possible mechanisms of regulation. METHODS: Alanine transport into confluent monolayers of endothelial cells was measured using 100 microM [3H]alanine in the presence and absence of external Na+, in cells deprived of serum for 24 hr (SD), and in SD cells exposed to 10% serum (S) for 3 hr (SD + S cells). RESULTS: Our results indicate that although SD did not significantly affect the Na(+)-independent transport of alanine when compared to normal cells, serum addition to serum-deprived cells markedly stimulated the Na(+)-dependent uptake of this amino acid through system A. The stimulation of alanine transport pathway(s) by serum was totally abolished by pretreatment of endothelial cells with 10 microM cycloheximide, suggesting a role of protein synthesis. Serum also induced a marked increase in calcium recycling at the cell membrane, suggesting that calcium is a key element of the serum signaling pathway. Indeed, both BAPTA (20 microM), a cellular calcium chelator, and thapsigargin (1 microM), an agent that depletes intracellular calcium stores, prevented the stimulation of alanine uptake by serum. Finally, pertussis toxin (400 ng/ml), an agent known to inactivate certain G-protein-dependent pathways, significantly reduced the serum-dependent 45Ca uptake and [3H]alanine entry. However, the protein kinase C activator PMA (100 nM), significantly reduced the stimulation of alanine uptake by serum but did not affect the stimulation of calcium uptake. CONCLUSIONS: Altogether these findings suggest that cell calcium is involved in the regulation of system A by serum in vascular endothelial cells.     

Preconditioning with bright light evokes a protective response against light damage in the rat retina

Constant exposure to bright light induces photoreceptor degeneration and at the same time upregulates the expression of several neurotrophic factors in the retina. At issue is whether the induced neurotrophic factors protect photoreceptors. We used a preconditioning paradigm to show that animals preconditioned with bright light became resistant to subsequent light damage. The preconditioning consisted of a 12-48 hr preexposure, followed by a 48 hr "rest phase" of normal cyclic lighting. The greatest protection was achieved by a 12 hr preexposure. Preconditioning induces a prolonged increase in two endogenous neurotrophic factors: basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). It also stimulates the phosphorylation of extracellular signal-regulated protein kinases (Erks) in both photoreceptors and Müller cells. These findings indicate that exposure to bright light initiates two opposing processes: a fast degenerative process that kills photoreceptors and a relatively slower process that leads to the protection of photoreceptors. The extent of light damage, therefore, depends on the interaction of the two processes. These results also suggest a role of endogenous bFGF and CNTF in photoreceptor protection and the importance of Erk activation in photoreceptor survival.     

Modulation of the tumor disposition of vinca alkaloids by PSC 833 in vitro and in vivo

PSC 833, a nonimmunosuppressive cyclosporin, is able to inhibit the efflux of antitumor drugs mediated by P-glycoprotein (P-gp). The purpose of the present study is to compare the effect of PSC 833 on the tumor disposition of [3H]vincristine ([3H]VCR) and [3H]vinblastine ([3H]VBL) in in vitro and in vivo experiments from a pharmacokinetic point of view. In in vitro experiments, the effect of PSC 833 was investigated on the cellular uptake of [3H]VCR and [3H]VBL by HCT-15 and COLO 205, human colorectal tumor cell lines with extensive and minimal expression of P-gp, respectively. PSC 833 (2 microM) increased the cellular uptake of [3H]VCR and [3H]VBL by HCT-15 cells, but not that by COLO 205 cells, 8- and 6-fold, respectively, without affecting the initial influx rates. In addition, 2 microM PSC 833 reduced the efflux of [3H]VCR from HCT-15 cells to a level comparable with that from COLO 205 cells. Furthermore, the effect of PSC 833 on the tumor disposition of intravenously administered [3H]VCR and [3H]VBL was studied in tumor inoculated mice. Infusion of PSC 833 (10 microg/hr/mouse) increased the HCT-15 tumor disposition of [3H]VBL and [3H]VCR in vivo to a level comparable with that observed in vitro. These findings demonstrate that PSC 833 enhances the tumor disposition of vinca alkaloids by inhibition of P-gp-mediated efflux not only in vitro but also in vivo in a solid tumor model.     

Metabolic studies of guinea pig basophilic leukocytes in short-term tissue culture. I. Measurement of histaminesynthesizing capacity by using an isotopic-thin layer chromatographic assay

Mature circulating guinea pig basophils, purified to comprise 25% or more of leukocytes, have been successfully maintained in short-term tissue culture for up to 72 hr. These cells were found to retain the ability to synthesize histamine, as assayed by a new isotopic-thin layer chromatographic assay which can reliably detect as little as 0.5 pg of 3H-histamine. Cell-associated, newly synthesized histamine was detectable as early as 1 hr of culture, was substantially increased at 6 hr, and reached maximal levels at 24 hr, when it accounted for approximately 6.5% of total cell histamine. Newly synthesized histamine was still detectable at 48 and 72 hr of culture. Histamine synthesis was decreased by lwoering the concentration of histidine in the culture medium, and was markedly reduced by all the specific histidine dedarboxylase (HDC)3 inhibitors tested, but not by alpha-methyl-DOPA, pyrilamine maleate, or metiamide. Increasing the concentration of pyridoxal phosphate, the HDC coenzyme, above that normally present in culture medium resulted in only an equivocal increase in the amount of newly synthesized histamine, whereas aminoguanidine, an inhibitor of histaminase, had no detectable effect. Uptake of exogenous histamine by cultured basophils was trivial compared to histamine synthesis. Both newly synthesized and previously manufactured, nonisotopic, histamine seemed to be stored in the same pool, as the same proportion of both was released by concanavalin A (Con A). Cellular histamine was largely conserved, with little or no spontaneous release into the medium of detectable isotopic or nonisotopic histamine. These techniques provide a model for studying granulocyte metabolic processes in vitro, and should assist in the direct investigation of a variety of their physiologic functions.     

Investigation of the presynaptic effects of quinine and quinidine on the release and uptake of monoamines in rat brain tissue

Quinine and quinidine are reported to potentiate the behavioural effects of serotonergic agents and monoamine uptake inhibitors. We have therefore investigated the presynaptic actions of quinine and quinidine on monoamine uptake and release in rat brain tissue in vitro. Quinidine evoked the release of [3H]5-HT, [3H]noradrenaline and [3H]dopamine from pre-loaded rat brain slices in a concentration dependent manner with EC50 values of 175, 486 and 150 microM, respectively. Quinine induced [3H]monoamine release with similar potencies. Both quinine and quinidine also inhibited the active uptake of [3H]5-HT, [3H]noradrenaline and [3H]dopamine into rat brain synaptosomes with IC50 values in the range 0.13-12.4 microM. The potency of each drug to inhibit [3H]5-HT uptake was significantly higher than that for [3H]noradrenaline or [3H]dopamine. The relative potency of quinidine compared to quinine was more marked in the case of [3H]5-HT (58-fold) than for [3H]noradrenaline (3-fold) or [3H]dopamine (4-fold). The inhibition of [3H]5-HT uptake by quinine and quinidine was competitive in nature and corresponded with the potencies of these drugs to inhibit [3H]paroxetine binding. No correlation was observed between the potencies of quinine and quinidine to induce the release of [3H]monoamines and to inhibit their uptake, suggesting that these effects are mediated by two distinct mechanisms. We conclude that the presynaptic actions of quinine and quinidine on monoamine uptake and release may be implicated in their potentiation of the effects of serotonergic agents and uptake blockers.     

Glia conditioned medium protects fetal rat midbrain neurones in culture from L-DOPA toxicity

L-DOPA kills dopamine neurones in culture but is the most effective drug for the treatment of Parkinson's disease, where it exhibits no clear toxicity. While glial cells surround and protect neurones in vivo, neurones are usually cultured in vitro in the absence of glia. We treated fetal midbrain rat neurones with L-DOPA, mesencephalic glia conditioned medium (CM) and L-DOPA + CM. L-DOPA reduced the number of tyrosine hydroxylase-positive (TH+) cells and [3H]DA uptake, and increased quinone levels. L-DOPA + CM restored [3H]DA uptake and quinone levels to normal, and increased the number of TH+ cells and terminals to 170% of control. CM greatly increased the number of TH+ cells and [3H]DA uptake. Mesencephalic glia therefore produced soluble factors which are neurotrophic for dopamine neurones, and which protect these neurones from the toxic effects of L-DOPA.     

Selection and retention of nurses

Secretory vesicles store neurotransmitters that are released by exocytosis. Their membrane contains transporters responsible for transmitter loading that are driven by an electrochemical proton gradient across the vesicle membrane. We have now examined whether uptake of noradrenaline is regulated by heterotrimeric G proteins. In streptolysin O-permeabilized PC 12 cells, GTP-analogues and AlF4- inhibited noradrenaline uptake, an effect that was sensitive to treatment with pertussis toxin. Inhibition of uptake was prevented by Galphao-specific antibodies and mimicked by purified activated Galphao2. No effect was seen when Galphao2 in its inactive GDP-bound form or purified activated Galphao1, Galphai1 and Galphai2 were tested. Down-regulation of uptake remained unchanged when exocytosis was inhibited by the light chain of tetanus toxin. Vesicular acidification was not affected whereas binding of [3H]reserpine was reduced by GTPgammaS and Galphao2. These data suggest that the monoamine transporter rather than the vacuolar ATPase is affected. We conclude that catecholamine uptake is controlled by Galphao2, suggesting a novel function for heterotrimeric G proteins in the control of neurotransmitter storage.     

U73122 and U73343 inhibit receptor-mediated phospholipase D activation downstream of phospholipase C in CHO cells

We investigated the effects of nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside on basal and K+-evoked release of [3H]noradrenaline from superfused synaptosomes from the rat cerebral cortex. Both substances produced concentration-dependent increases in the release of the labeled transmitter under basal and depolarized conditions. The effects of the donors on basal release were Ca2+-independent but were not inhibited by the carrier-uptake blocker, desipramine; the effects were abolished by hemoglobin (an NO scavenger). Thirty-five minutes after stimulation with sodium nitroprusside, the synaptosomes were still responsive to KCl stimulation, indicating that the donor's effects were not caused by damage to the synaptosome membrane. The cGMP analogue, 8-bromo-cGMP, had no effect on basal release, and the enhanced release produced by sodium nitroprusside was not inhibited by the specific inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, indicating that NO's effects on basal release of the neurotransmitter are guanylate cyclase-independent. Both of the NO donors had more marked effects on release of [3H]noradrenaline during K+-stimulated depolarization. The NO-mediated increase in this case was partially antagonized by 10 microM LH-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, and 8-Br-cGMP was also capable of producing concentration-dependent increases in the K+-stimulated release of the transmitter. These findings indicate that the effects of the NO donors on [3H]noradrenaline release during depolarization are partially mediated by the activation of guanylate cyclase.     

Inhibition of nuclear uptake of glucocorticoid-receptor complex by aurintricarboxylic acid

The presence of 50-200 microM aurintricarboxylic acid (ATA) blocked the uptake of [3H]-triamcinolone acetonide (3H-TA)-receptor complex from rat liver cytosol by isolated nuclei. The half-maximal inhibition (I.D.50) in the nuclear uptake of [3H]-TA-receptor complex was observed at 70- and 80 microM ATA depending upon whether the inhibitor was added prior to or following receptor activation. In addition, the nuclear-bound [3H]-TA-receptor complex from control samples could be completely extracted by an incubation with 20-100 microM ATA. The amount of [3H]-TA-receptor complex remained unchanged under these conditions. The effects of ATA may result due to its interaction with the glucocorticoid receptor at/near the sites that are involved in its nuclear uptake. ATA, therefore is a potentially useful chemical probe for analysis of glucocorticoid receptor.     

Pentobarbital enhances cyclic adenosine monophosphate production in the brain by effects on neurons but not glia

BACKGROUND: Cyclic adenosine monophosphate (cAMP) is an important regulator of neuronal excitability. The effects of barbiturates on cAMP production in intact neurons are not known. This study used cultures of cortical neurons, cultures of glia, and slices of cerebral cortex from the rat to study the effects of barbiturates on cAMP regulation in the brain. METHODS: Primary cultures of cortical neurons or glia were prepared from 17-day gestational Sprague-Dawley rat fetuses and were used after 12-16 days in culture. Cross-cut slices (300 microns) were prepared from cerebral cortex of adult rats. Cyclic AMP accumulation was determined by measuring the conversion of [3H]adenosine triphosphate (ATP) to [3H]cAMP in cells preloaded with [3H]adenine. RESULTS: Pentobarbital enhanced isoproterenol- and forskolin-stimulated, but not basal, cAMP accumulation in cultures of cerebral neurons. Cyclic AMP production was enhanced by pentobarbital in a dose-dependent fashion up to a concentration of 250 microM; This concentration of pentobarbital increased cAMP production by 40-50% relative to that in controls without pentobarbital. At 500 microM pentobarbital, the magnitude of the enhancement was less. Pentobarbital had no effect on isoproterenol-stimulated cAMP production in cultures containing only glia. Pentobarbital also enhanced isoproterenol-stimulated, but not basal, cAMP production in slices of cerebral cortex by approximately 30% at concentrations of 62.5-250 microM and by almost 100% at 500 microM. CONCLUSIONS: Pentobarbital enhances stimulated cAMP accumulation in cultured preparations from brain and fresh cortical slices. Neurons are required for this effect. Because cAMP modulates neuronal excitability, this effect of pentobarbital may be an important mechanism by which this anesthetic influences brain function.     

Selectivity of hemicholinium mustard, an affinity ligand, for the high-affinity choline transport system

The selectivity of the irreversible inhibition of high-affinity choline uptake (HACU) by hemicholinium mustard (HCM; 2,2'-(4,4'-biphenylene)bis[2-hydroxy-4-(2-bromoethyl)-morpholine] hydrochloride) with respect to other cholinergic proteins and other sodium-dependent transport systems was examined. Preincubation of rat forebrain membranes with HCM, followed by washing and measurement of [3H]-hemicholinium-3 binding to the high-affinity choline transporter, was shown to decrease binding capacity (Bmax) by 70% without affecting the apparent affinity of the ligand. However, a similar preincubation, wash and binding experiment using [3H]-NMS as a ligand for muscarinic receptors showed no HCM effect on binding parameters. To measure the effects of HCM on choline acetyltransferase (ChAT), synaptosomes were incubated in HCM, then washed. The synaptosomes were lysed and ChAT activity was measured. Treatment with 50 microM HCM, a concentration that inhibits 100% of synaptosomal HACU, results in a 24% decrease in ChAT activity. HCM demonstrates little residual inhibition of other sodium-dependent neurotransmitter transporter transporters: preincubation with 50 microM HCM results in a decrease of 12% in transport of [3H]-dopamine and a decrease of 6% in the transport of [3H]-GABA. The binding of HCM, like that of hemicholinium-3 is sodium-dependent. HCM preincubation in the presence of sodium results in inhibition of HACU to 32% of control; in the absence of sodium HACU is 65% of control. This represents a loss of 51% of the observed irreversible inhibition produced by HCM. Irreversible inhibition by HCM can also be prevented by co-incubation with hemicholinium-3. Co-incubation with hemicholinium-3 results in residual HACU inhibition that decreases from 51% (HCM alone) to 28% (HCM + hemicholinium-3). When atropine instead of hemicholinium-3 is co-incubated with HCM, HCM still inhibits 40% of transport, demonstrating the pharmacological specificity of the protective effect of hemicholinium-3. Experiments in the guinea-pig myenteric plexus preparation demonstrate a gradual recovery from the residual effects of HCM. Evoked ACh release decreases to 24% immediately following treatment with 1 microM HCM. After 2 hr of recovery, tissues have recovered to about 50% of control levels, after which recovery continues at a slower rate.     

Synaptic vesicle recycling in cultured cerebellar granule cells: role of vesicular acidification and refilling

The role of the transvesicular protonmotive force in synaptic vesicle recycling was investigated in cultured cerebellar granule cells. The vesicular V-ATPase was inhibited by 1 microM bafilomycin A1; as an alternative, the pH component of the gradient was selectively collapsed by equilibration of the cells with 10 mM methylamine and monitored with the fluorescent probe Lysosensor Green. Electrical field-evoked exocytosis of D-[3H]aspartate was inhibited by bafilomycin A1 but not by methylamine, indicating that a transvesicular membrane potential rather than pH gradient is required for transmitter retention within vesicles. In contrast, neither compound affected the field-evoked uptake, recycling, or destaining of the vesicle-specific dye FM2-10; thus, vesicles whose lumens were neutral and/or depleted of transmitter could still recycle in the nerve terminal. No exhaustion of D-[3H]aspartate exocytosis was observed when cells were subjected to six consecutive trains of field stimuli (40 Hz/10 s separated by 10 s). In contrast, the release of preloaded FM2-10 was reduced by approximately 50%, with each stimulus indicating that unlabeled vesicles with accumulated D-[3H]aspartate were competing with labeled vesicles for exocytosis. As D-[3H]aspartate was accumulated rapidly across the vesicle membrane from the large cytoplasmic pool, the transmitter-loaded but unlabelled vesicles may represent refilled recycling vesicles. FM2-10 destaining and D-[3H]aspartate exocytosis were reduced in parallel at low frequencies, challenging a role for transient vesicle fusion.     

Presynaptic cholinergic actions by the putative cognitive enhancing agent DuP 996

3,3-Bis(4-pyridinylmethyl)-1-phenylindolin-2-one (DuP 996), an agent that improves the performance of rodents on memory-related behavioral tasks, was tested in rat hippocampal synaptosomes to evaluate putative direct central nervous system cholinoregulatory actions. At low micromolar concentrations, DuP 996 enhanced stimulated [3H]acetylcholine ([3H]ACh) release in a manner that was markedly dependent upon experimental test conditions. For tissues exposed to potassium-enriched buffer (9.4-40 mM KCl) or calcium-enriched buffer (for calcium-naive synaptosomes), DuP 996 facilitated [3H]ACh release (EC50 = 0.5 microM) only when extracellular potassium was moderately elevated (15-22.5 mM); at lower or higher potassium concentrations, DuP 996 failed to influence calcium-dependent transmitter release. However, under conditions where DuP 996 was maximally effective, there was no apparent change in presynaptic inhibition mediated through muscarinic autoreceptors. In contrast, when synaptosomes were depolarized before drug infusion (S2/S1 paradigm), the facilitatory action of DuP 996 was approximately 10-fold less potent and exhibited no dependence on extracellular potassium concentration. Relevant to the latter observation, it was noted that brief treatment with DuP 996 produced a long-lasting activation of synaptosomal high-affinity [3H]choline uptake that was completely calcium dependent and additive with high potassium. Taken together, these results indicate that DuP 996 exerts two direct but possibly distinct actions on hippocampal cholinergic nerve endings. First, this drug facilitates stimulus-dependent release of ACh through a mechanism or site that appears to be influenced by the neuronal membrane potential. In addition, DuP 996 enhances ACh synthesis and, thereby, may help replenish releasable stores of transmitter and maintain synaptic transmission during periods of intense (repetitive) neuronal firing.     

Determination of pindolol enantiomers in human plasma and urine by simple liquid-liquid extraction and high-performance liquid chromatography

Ethanol disrupts signal transduction mediated by a variety of G-protein coupled receptors. We examined the effects of ethanol on arachidonic acid release mediated by muscarinic acetylcholine receptors. Chinese hamster ovary (CHO) cells transfected with the different subtypes of human muscarinic receptors (M1 to M5) were incubated with [3H]arachidonic acid ([3H]AA) for 18 hr, washed, and exposed to the cholinergic agonist carbamylcholine for 15 min. Carbamylcholine induced [3H]AA release from CHO cells expressing M1, M3, or M5, but not M2 or M4, muscarinic receptors. Dose response curves revealed that carbamylcholine stimulated [3H]AA release by up to 12-fold with an ECo of approximately 0.4 microM; maximal responses were obtained with 10 microM carbamylcholine. Exposure of M1-, M3-, or M5-expressing cells to ethanol for 5 min before stimulating with carbamylcholine reduced [3H]AA release by 40 to 65%; 50% of the maximal inhibition was obtained with an ethanol concentration of 30 to 50 mM. Ethanol did not affect basal [3H]AA release measured in the absence of carbamylcholine. Dose response curves suggest that ethanol acts as a noncompetitive inhibitor of muscarinic receptor-induced [3H]AA release insofar as maximal [3H]AA release was depressed in the presence of ethanol with no apparent change in the EC50 for stimulation by carbamylcholine. Exposure of CHO cells to 38 mM ethanol for 48 hr increased [3H]AA release induced by carbamylcholine without affecting basal [3H]AA release or altering the EC50 for carbamylcholine. These results indicate that ethanol acutely inhibits muscarinic receptor signaling through the arachidonic acid pathway in a noncompetitive manner, but chronically enhances muscarinic signaling through the same pathway.