Organic calcium channel blockers enhance [3H]purine release from rat brain cortical synaptosomes

The release of [³H]purines was investigated in a crude mitochondrial fraction (P₂ fraction) from rat brain cortex pre-loaded with [³H]adenosine for 30 sec at 37°C in vitro. Potassium, veratridine and glutamate were used as depolarizing agents to evoke the release of [³H]purines. Ca²⁺ removal, the addition of EGTA, and treatment with organic or inorganic Ca²⁺ antagonists did not inhibit [³H]purine release in this preparation. On the other hand, Ca²⁺ removal and the addition of EGTA greatly enhanced³H-purine release induced by glutamate. D-600 and diltiazem enhanced K⁺-evoked [³H]purine release, and nifedipine increased veratridine evoked [³H]purine release indicating that either these Ca²⁺ antagonists have different sites of action, or that K⁺ and veratridine may release [³H]purine from different metabolic pools. Organic Ca²⁺ antagonists failed to enhance the [³H]purine release evoked by glutamate, further supporting the notion that various depolarizing agents may release [³H]purines from different cellular compartments.     

Characterization of carrier-mediated efflux in human embryonic kidney 293 cells stably expressing the rat serotonin transporter: a superfusion study

Human embryonic kidney 293 cells stably transfected with the rat plasmalemmal serotonin transporter (rSERT) were incubated with 5-[3H]hydroxytryptamine ([3H]5-HT) and superfused. Substrates of the rSERT, such as p-chloroamphetamine (PCA) or methylenedioxymethamphetamine, concentration-dependently increased basal efflux of [3H]5-HT. 5-HT reuptake blockers (e.g., imipramine, citalopram) also caused an enhancement of [3H]5-HT efflux, reaching about half the maximal effect of the rSERT substrates. In uptake experiments, both groups of substances concentration-dependently inhibited 5-HT uptake. EC50 values obtained in superfusion experiments significantly correlated with IC50 values from uptake studies (r2 = 0.92). Addition of the Na+,K(+)-ATPase inhibitor ouabain (100 microM) to or the omission of K+ from the superfusion buffer accelerated basal efflux. The effect of PCA (10 microM) was markedly enhanced by both measures, whereas the effect of uptake inhibitors remained unchanged. When [3H]MPP+, a substrate with low affinity for the rSERT, was used instead of [3H]5-HT for labeling the cells, uptake inhibitors failed to augment efflux. By contrast, PCA accelerated [3H]MPP+ efflux, and its effect was strongly enhanced in the presence of ouabain. The results suggest that the [3H]5-HT efflux caused by substrates of rSERT is carrier-mediated, whereas efflux induced by uptake inhibitors is a consequence of interrupted high-affinity reuptake that is ongoing even under superfusion conditions.     

Interaction Between A1 Adenosine and Class II Metabotropic Glutamate Receptors in the Regulation of Purine and Glutamate Release from Rat Hippocampal Slices

Abstract: Electrical stimulation of rat hippocampal slices evoked the release of excitatory amino acids and purines, as reflected by a time-dependent increase in the extracellular levels of glutamate and adenosine, as well as by the increased efflux of radioactivity in slices preloaded with both [¹⁴C]glutamate and [³H]adenosine. The evoked release of excitatory amino acids and purines was amplified when slices were exposed to 8-cyclopentyl-1,3-dipropylxanthine (a selective A1 adenosine receptor antagonist), (+)-α-methyl-4-carboxyphenylglycine [a mixed antagonist of metabotropic glutamate receptors (mGluRs)], or (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (a selective antagonist of class II mGluRs). In contrast, 2-chloro-N⁶-cyclopentyladenosine (CCPA; a selective A1 receptor agonist) or (2S,1R,2R,3R)-(2,3-dicarboxycyclopropyl)glycine (DCG-IV; a selective agonist of class II mGluRs) reduced the evoked release of excitatory amino acids and purines. CCPA and DCG-IV also reduced the increase in cyclic AMP formation induced by either forskolin or electrical stimulation in hippocampal slices. The inhibitory effect of CCPA and DCG-IV on release or cyclic AMP formation was less than additive. We conclude that the evoked release of excitatory amino acids and purines is under an inhibitory control by A1 receptors and class II mGluRs, i.e., mGluR2 or 3, which appear to operate through a common transduction pathway. In addition, although these receptors are activated by endogenous adenosine and glutamate, they can still respond to pharmacological agonists. This provides a rationale for the use of A1 or class II mGluR agonists as neuroprotective agents in experimental models of excitotoxic neuronal degeneration.     

CB1-cannabinoid receptors are involved in the modulation of non-synaptic [3H]serotonin release from the rat hippocampus

In the present study we investigated whether serotonin release in the hippocampus is subject to regulation via cannabinoid receptors. Both rat and mouse hippocampal slices were preincubated with [3H]serotonin ([3H]5-HT) and superfused with medium containing serotonin reuptake inhibitor citalopram hydrobromide (300 nM). The cannabinoid receptor agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate (WIN55,212-2, 1 microM) did not affect either the resting or the electrically evoked [3H]5-HT release. In the presence of the ionotropic glutamate receptor antagonists D(-)-2-amino-5-phosphonopentanoic acid (AP-5, 50 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione-disodium (CNQX, 10 microM) the evoked [3H]5-HT release was decreased significantly. Similar findings were obtained when CNQX (10 microM) was applied alone with WIN55,212-2. This effect was abolished by the selective cannabinoid receptor subtype 1 (CB1) antagonists N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716, 1 microM) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide trifluoroacetate salt (AM251, 1 microM). Similarly to that observed in rats, WIN55,212-2 (1 microM) decreased the evoked [3H]5-HT efflux in wild-type mice (CB1+/+). The inhibitory effect of WIN55,212-2 (1 microM) was completely absent in hippocampal slices derived from mice genetically deficient in CB1 cannabinoid receptors (CB1-/-). Relatively selective degeneration of fine serotonergic axons by the neurotoxin parachloramphetamine (PCA) reduced significantly the tritium uptake and the evoked [3H]5-HT release. In addition, PCA, eliminated the effect of WIN55,212-2 (1 microM) on the stimulation-evoked [3H]5-HT efflux. In contrast to the PCA-treated animals, WIN55,212-2 (1 microM) reduced the [3H]5-HT efflux in the saline-treated group. Our data suggest that a subpopulation of non-synaptic serotonergic afferents express CB1 receptors and activation of these CB1 receptors leads to a decrease in 5-HT release.     

Biosynthesis and scavenging of purines by pathogenic mycobacteria including Mycobacterium leprae

Purine biosynthesis de novo could not be detected in suspensions of Mycobacterium leprae isolated from armadillo tissue. In contrast, non-growing suspensions of other pathogenic mycobacteria, also isolated from infected host tissue did synthesize purines. Rates of synthesis, judged by incorporation of [2-14C]glycine or [3-14C]serine into nucleic acid purines were 600 times higher in M. microti and 110 times higher in M. avium--both isolated from infected mouse tissue--than the lowest possible rate detectable and therefore the highest possible rate in M. leprae. The rate of purine synthesis relative to purine scavenging (judged by comparing incorporation of [3-14C]serine and [8-14C]hypoxanthine into nucleic acid purines in suspensions of mycobacteria) varied only slightly--4-fold in M. microti and 6-fold in M. avium--whether organisms were harvested from media with or without purines, from media with a low nitrogen content but containing a purine, from mice or even with starved organisms. Thus, the failure of M. leprae to synthesize purines could not be explained as either a result of using non-growing mycobacteria in the incubations with 14C-labelled precursors or as repression or inhibition of synthesis de novo. It appears that M. leprae requires a supply of the purine ring from its environment. Nucleotides, which may be the major source of the purine ring in the intracellular environment, were not taken up directly by M. leprae but could be hydrolysed first to nucleosides and then taken up.     

Brain-to-blood efflux transport of estrone-3-sulfate at the blood-brain barrier in rats

Efflux transport of estrogens such as estrone-3-sulfate (E1S), and estrone (E1) across the blood-brain barrier (BBB) was evaluated using the Brain Efflux Index (BEI) method. The apparent BBB efflux rate constant (Keff) of [3H]E1S, and [3H]E1 was 6.63 x 10(-2) +/- 0.77 x 10(-2) min(-1), and 6.91 x 10(-2) +/- 1.23 x 10(-2) min(-1), respectively. The efflux transport of [3H]E1S from brain across the BBB was a saturable process with Michaelis constant (Km) of 96.0 +/- 34.4 microM and 93.4 +/- 22.0 microM estimated by two different methods. By determining [3H]E1S metabolites using high performance liquid chromatography (HPLC) after intracerebral injection, significant amounts of [3H]E1S were found in the jugular venous plasma, providing direct evidence that most of [3H]E1S is transported from brain across the BBB in intact form. To compare the apparent efflux clearance across the BBB of E1S with that of E1, the brain distribution volume of E1S and E1 was estimated using the brain slice uptake method. The apparent efflux clearance of [3H]E1S was determined to be 74.9 +/- 3.8 microl/(min x g brain) due to the distribution volume of 1.13 +/- 0.06 ml/g brain. By contrast, the apparent efflux clearance of E1 was more than 227 +/- 3 microl/(min x g brain), since the distribution volume of [3H]E1 at 60 min was 3.28 +/- 0.13 ml/g. The E1S efflux transport process was inhibited by more than 40% by coadministration of bile acids (taurocholate, and cholate), and organic anions (sulfobromophthalein, and probenecid), whereas other organic anions did not affect the E1S efflux transport. The [3H]E1S efflux was significantly reduced by 48.6% after preadministration of 5 mM dehydroepiandrosterone sulfate. These results suggest that E1S is transported from brain to the circulating blood across the BBB via a carrier-mediated efflux transport system.     

Muscarinic Modulation of Purine Release from Electrically Stimulated Rat Cortical Slices

The release of 3H-labeled purines at rest and during electrical stimulation was investigated in slices of rat cortex prelabeled with [3H]adenine and perfused with Krebs solution. A linear relationship was found between radioactivity efflux and stimulation frequency from 2.5 to 20 Hz. At frequencies of less than 2.5 Hz, no increase in radioactivity efflux was detected. The amount of tritium released per pulse increased with stimulation frequency up to 10 Hz and declined at 20 Hz. The tritium efflux from the slices at rest and at a stimulation frequency of 10 Hz, analyzed by HPLC with ultraviolet absorbance detection at 254 nm, consisted mostly of adenosine, inosine, and hypoxanthine. The 3H-labeled purine release evoked by 10-Hz stimulation increased with current intensity from 15 to 100 mA/cm2. At 20 mA/cm2, addition of 0.5 microM tetrodotoxin to the superfusing Krebs solution brought about a 98% decrease of 3H-labeled purine release. At higher current strength, the percentage of tetrodotoxin-sensitive-evoked tritium efflux was smaller. At 30 mA/cm2, 86% of the evoked release was tetrodotoxin sensitive. Under these stimulation conditions, tritium efflux showed a 69% decrease when the slices were superfused with calcium-free Krebs solution containing 0.5 mM EGTA. The muscarinic agonist oxotremorine (30 microM) significantly enhanced the 10-Hz-stimulated 3H-labeled purine release. The effect of oxotremorine was partially prevented by tetrodotoxin, was antagonized by atropine (1.5 microM), and was mimicked by addition of physostigmine (3.8 microM) to the superfusion fluid. Atropine alone did not affect the evoked release, and none of the drugs modified the basal tritium efflux.(ABSTRACT TRUNCATED AT 250 WORDS)     

Striatal Dopamine Release In Vitro: A β-Adrenoceptor-Regulated Response Not Mediated Through Cyclic AMP

The effects of (-)isoproterenol (10(-6) M), dibutyryl cyclic AMP (10(-3) M), and the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) (10(-4) M) on in vitro [3H]dopamine ([3H]DA) efflux and synthesis were studied in rat striatal slices continuously superfused with [3H]tyrosine. The beta-adrenoceptor agonist (-)isoproterenol induced an immediate and significant facilitation of [3H]DA efflux but did not alter [3H]DA synthesis as measured by [3H]H2O formation. In contrast, both dibutyryl cyclic AMP and IBMX enhanced [3H]DA synthesis as well as efflux. The presence of IBMX in the superfusing medium did not potentiate the augmentation of [3H]DA efflux caused by (-)isoproterenol. Additionally, the blockade of [3H]DA synthesis by alpha-methyl-p-tyrosine (10(-4) M) completely prevented the action of dibutyryl cyclic AMP on [3H]DA efflux. However, under similar conditions, (-)isoproterenol was still able to increase [3H]DA efflux. The results suggest that (-)isoproterenol can modify striatal DA release through a mechanism not involving cyclic AMP.     

Effects of docosahexaenoic and arachidonic acids on the synthesis and distribution of aminophospholipids during neuronal differentiation of PC12 cells

We have shown previously that docosahexaenoic acid (DHA) promotes and arachidonic acid (AA) suppresses neurite outgrowth of PC12 cells induced by nerve growth factor (NGF) and that incorporation of [3H]ethanolamine into phosphatidylethanolamine (PE) is suppressed in PC12 cells by AA while DHA has no effect. In the present study, the effects of these fatty acids on PE synthesis via decarboxylation of phosphatidylserine (PS), another pathway of PE synthesis, and distribution of aminophospholipids were examined. Incorporation of [3H]serine into PS and PE was elevated in the course of NGF-induced differentiation and was further stimulated significantly by DHA, but not by AA. [3H]Ethanolamine uptake by PC12 cells was significantly suppressed by AA but not by DHA while these fatty acids did not affect [3H]serine uptake, indicating that the suppression by AA of [3H]ethanolamine incorporation into phosphatidylethanolamine is attributable, at least in part, to a reduction in [3H]ethanolamine uptake. The distribution of PE in the outer leaflet of plasma membrane decreased during differentiation, which is known to be accompanied by an increase in the surface area of plasma membrane. Supplementation of PC12 cells with DHA or AA did not affect the distribution of aminophospholipids. Thus, DHA and AA affected aminophospholipid synthesis and neurite outgrowth differently, but not the transport and distribution of aminophospholipids, while the PE concentration in the outer leaflet of the plasma membrane decreased in association with morphological changes in PC12 cells induced by NGF.     

Brain Purines in a Genetic Mouse Model of Lesch-Nyhan Disease

Abstract: Mice carrying a mutation in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) have recently been produced to provide an animal model for Lesch-Nyhan disease. The current-studies were conducted to characterize the consequences of the mutation on the expression of HPRT and to characterize potential changes in brain purine content in these mutants. Our results indicate that the mutant animals have no detectable HPRT-immunoreactive material on western blots and no detectable HPRT enzyme activity in brain tissue homogenates, confirming that they are completely HPRT deficient (HPRT^-). Despite the absence of HPRT-mediated purine salvage, the animals have apparently normal brain purine content. However, de novo purine synthesis, as measured by [¹⁴C]formate incorporation into brain purines, is accelerated four- to fivefold in the mutant animals. This increase in the synthesis of purines may protect the HPRT^- mice from potential depletion of brain purines despite complete impairment of HPRT-mediated purine salvage.     

Hypoxanthine Transport and Metabolism in the Central Nervous System

The mechanisms by which hypoxanthine, the principal purine in plasma and CSF, enters and leaves rabbit brain, choroid plexus, and CSF were investigated in the isolated choroid plexus in vitro and by injecting [14C]hypoxanthine intraventricularly and [3H]hypoxanthine intravenously. The isolated choroid plexus accumulated and extensively metabolized [14C]hypoxanthine; however, 14C was readily released from choroid plexus principally as [14C]-hypoxanthine. After infusion of [3H]hypoxanthine intravenously, [3H]hypoxanthine entered CSF and brain slowly and was converted in brain to nucleotides. Fewer than 5% of the acid-soluble purine nucleotides in brain entered rabbit brain from plasma hypoxanthine (and inosine) per 24 h. After intraventricular injection of [14C]hypoxanthine, the [14C]hypoxanthine was cleared from the CSF into the blood or accumulated by brain and largely converted into 14C-nucleotides. Little [14C]xanthine and no [14C]uric acid or allantoin were formed. These studies show that brain, unlike most other tissues, rapidly recycles hypoxanthine and converts it into purine nucleotides, and not unsalvageable purines.     

Release of Endogenous and Accumulated Exogenous Amino Acids from Slices of Normal and Climbing Fibre-Deprived Rat Cerebellar Slices

Efflux of various amino acids from slices of rat cerebellar hemispheres was determined under resting or depolarizing conditions. It was increased under high K+(50 mM) as compared to low K+ (5 mM) conditions by 1258 pmol/mg protein for aspartate, 478 for gamma-aminobutyric acid (GABA), 44,693 for glutamate, and 615 for glycine. These were significantly higher than the corresponding values obtained under low-Ca2+ (0.1 mM), high-Mg2+ (12 mM) conditions, whereas for 11 other amino acids the K+-induced efflux was similar under normal and low-Ca2+ concentrations. The K+-induced efflux of exogenously accumulated L-[3H]aspartate, D-[3H]aspartate, and L-[3H]glutamate was higher by factors of 2, 5.8, and 6.3, respectively, under normal Ca2+ conditions, as compared with low-Ca2+, high-Mg2+ conditions. After climbing fibre degeneration induced by destruction of the inferior olive with 3-acetylpyridine, release of endogenous aspartate and exogenous L-[3H]glutamate and D-[3H]aspartate was significantly reduced, by 26%, 38%, and 27%, respectively. These results support the hypothesis that climbing fibres may use aspartate or a related compound as a neurotransmitter. In rat cerebellar tissue, L-[3H]glutamate and L-[3H]aspartate differ in several aspects: (1) L-[3H]glutamate uptake was 4 times higher than that of L-[3H]aspartate; (2) fractional rate constant of K+- evoked release of L-[3H]aspartate was 7% X 2.5 min-1, and of L-[3H]glutamate 36% X 2.5 min-1; and (3) specific activity of L-[3H]glutamate in the eluate collected during K+ stimulation was 3.5 times the value in the tissue, whereas for L-[3H]aspartate, specific activities in the eluate and tissue were similar.     

Activation of glutamate receptors promotes a calcium-dependent and transporter-mediated release of purines in cultured avian retinal cells: possible involvement of calcium/calmodulin-dependent protein kinase II

Calcium-dependent release of purines was previously demonstrated in cultures of chick retinal cells stimulated with high potassium concentrations but there is no evidence for an exocytotic mechanism of adenosine release from presynaptic terminals. Here we show that activation of NMDA or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate glutamate ionotropic receptors promotes a two- to three-fold increase in the release of purines from these cultures. Approximately 96% of intracellular radioactivity is found as nucleotides after incubation with [(3)H]adenosine, but more than 85% of glutamate-stimulated released material is found as inosine (60%), hypoxanthine (19.9%) and adenosine (7.8%). The release is prevented by removal of extracellular calcium, by the transporter blocker nitrobenzylthioinosine, or inhibitors of calcium/calmodulin-dependent protein kinase II (CAMK II). The uptake of [(3)H]adenosine, but not of [(3)H]GABA or [(3)H]choline, is also blocked by 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN62), N-[2-(N-(4-chlorocinnamyl)-N-methylaminomethyl)phenyl-N-[2-hydroxiethyl]-4-methoxybenzenesulfonamide (KN93) or the myristoylated autocamtide-2-related inhibitory peptide, suggesting that the enzyme modulates the nucleoside transporter. The distribution of intracellular purines was not affected by KN62. These results indicate that activation of glutamate receptors triggers the release of purines from retinal cells by a mechanism involving calcium influx, CAMK II and the nitrobenzylthioinosine-sensitive nucleoside transporter. The regulation of adenosine release by glutamate receptors and CAMK II could have important consequences in the presynaptic control of glutamate release.     

The efflux of biotin from human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PMBCs) are readily available for sampling and are a useful model for studying biotin metabolism in human cells. To better understand biotin handling by PMBCs, we investigated the mechanism(s) and kinetics of biotin efflux from PMBCs. Human PMBCs were incubated with [(3)H]biotin at 475 pmol/L to load the cells. The [(3)H]biotin-loaded cells were then harvested and incubated in [(3)H]biotin-free media for up to 20 hours. At various intervals, aliquots of the PMBC suspensions were collected and analyzed for intracellular [(3)H]biotin. [(3)H]Biotin efflux from cells at 37 degrees C was fast and triphasic; the half-lives for the three elimination phases were 0.2 +/- 0.02 hours, 1.2 +/- 0.1 hours, and 21.9 +/- 13.6 hours. Such a triphasic [(3)H]biotin efflux could reflect (1) rapid efflux of free biotin, (2) slower release of biotin bound to intracellular molecules, and (3) even slower release from carboxylases in cellular organelles. Incubation at 4 degrees C rather than 37 degrees C increased the [(3)H]biotin retained at 20 hours from 27% to 85%. This observation is consistent with transporter-mediated efflux. When cellular glucose utilization was reduced by 2-deoxy-d-glucose and sodium fluoride, [(3)H]biotin efflux was similar to controls, suggesting that biotin efflux does not directly require metabolic energy. When [(3)H]biotin-loaded cells were incubated in external medium containing unlabeled biotin analogs, [(3)H]biotin efflux was accelerated approximately two times compared with incubation in a biotin-free medium. This observation suggests that biotin efflux is mediated by the same transporter that mediates biotin uptake from the extracellular medium (i.e., classic countertransport).     

The efflux of l-carnitine from cells in culture (CCL 27)

The efflux of l-[³H]carnitine was studied in cells from an established cell line from human heart (Girardi human heart cells, CCL 27). The cells were loaded with 4 μmol/l l-[³H]carnitine for 1 or 24 h, and the efflux of radioactivity into the medium was measured. The amount of intracellular l-[³H]carnitine retained was expressed as a function of time. The results were fitted to an exponential equation, from which efflux rate constants were computed.Increasing the extracellular concentration of butyrobetaine, l-carnitine, d-carnitine, betaine, dl-norcarnitine or 3-dimethylamino-2-hydroxypropionic acid each increased the observed efflux. This is most likely due to accelerated exchange diffusion. The substrate specificity of this accelerated exchange diffusion is different from what previously has been found in competitive uptake studies of l-carnitine. l-Carnitine was preferentially released to l-acetylcarnitine, and blocking the sulfhydryl groups with 5,5-dithiobis(2-nitrobenzoic acid) increased the efflux.     

Comparison of [3H]Choline and d-[3H]Aspartate Efflux from Guinea Pig and Human Neocortex

The outflow of [3H]choline ([3H]Ch) evoked by electrical field stimulation and the efflux of D-[3H]Asp induced by 35 mM KCl and 1-10 microM ouabain were studied in human and guinea pig cortical slices, kept under identical experimental conditions. [3H]Ch outflow was significantly lower whereas D-[3H]Asp efflux was significantly higher in humans than in guinea pigs. This suggests a different proportion of the two neuronal systems in these two species. Blockade of muscarinic autoreceptors with atropine increased, whereas stimulation of alpha 2 receptors with norepinephrine (NE) reduced, the evoked [3H]Ch outflow to the same extent in human and guinea pig cortical slices. Conversely, NE did not affect ouabain-induced D-[3H]Asp efflux, suggesting that an alpha 2-mediated control is not operative in the glutamatergic cortical structures. Desmethylimipramine, 2-5 microM, was able to increase [3H]Ch outflow through atropine-like mechanisms only in the human. This drug at 20-50 microM inhibited [3H]Ch and D-[3H]Asp efflux in both species, through mechanisms unrelated to its monoamine reuptake blocking properties. Thus, similarities and differences can be detected between humans and guinea pigs with regard to (a) the relative potency of the cholinergic and acidic amino acidergic signals and (b) the modulation of neurotransmitter outflow by drugs acting on auto- and the heteroreceptors.     

Energy-dependent efflux of methotrexate in L1210 leukemia cells. Evidence for the role of an ATPase obtained with inside-out plasma membrane vesicles.

Earlier studies from our laboratory (Dembo, M., Sirotnak F. M., and Moccio, D. M. (1984) J. Membr. Biol. 78, 9-17) suggested that methotrexate (MTX) efflux from L1210 cells was mediated predominantly by an ATP-dependent, outwardly directed, mechanism. To examine this process further, we utilized predominantly (74%) inside-out plasma membrane vesicle preparations derived from an L1210 cell variant (L1210/R24) with 15-fold reduced Vmax for [3H]MTX influx. Efflux of [3H]MTX, under nonionic buffer conditions, in these inside-out membrane vesicles was temperature and ATP dependent (apparent Km = 0.40 +/- 0.06 mM), osmotically sensitive, and unaffected by protonophores. The presence of K+, Na+, Cl-, and HCO3- at their physiological concentrations had no effect on [3H]MTX efflux. Other triphosphonucleotides (GTP and CTP), but not a nonhydrolyzable analogue, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), could also stimulate efflux, but to a lesser extent. Also, ATP gamma S and orthovanadate were potent inhibitors of ATP-dependent efflux of [3H]MTX. Other experiments revealed a system with low saturability for [3H]MTX during efflux (apparent Km = 46 +/- 7 microM), but extremely high capacity (106 +/- 15 pmol/min/mg protein), and a pH optimum in the range of 5.5-6. However, appreciable efflux was measured in the physiological range of pH 6.7-6.9. A number of inhibitors or copermeants for ATP-dependent [3H]MTX efflux in intact L1210 cells were inhibitors of ATP-dependent efflux in inside-out plasma membrane vesicles, including, cholate, bromosulfophthalein, verapamil, quinidine, and reserpine. These findings and other results showing that bromosulfophthalein will completely inhibit efflux are consistent with a role for an ATPase in [3H]MTX efflux, and suggest that the process under study is the bromosulfophthalein-sensitive, ATP-dependent route responsible for the majority of [3H]MTX efflux in intact L1210 cells.     

Purine nucleoside-mediated protection of chemical hypoxia-induced neuronal injuries involves p42/44 MAPK activation

Hypoxia in brain may lead to cell death by apoptosis and necrosis. Concomitant is the formation of purine nucleosides, e.g. adenosine, a powerful endogenous neuroprotectant. Despite vigorous studies, many aspects of the mechanisms involved in purine-based protection are still unclear. In this study, we wanted to investigate the effect of purine nucleosides on cellular responses to chemical hypoxia. O(2)-sensitive neuronal pheochromocytoma (PC12)-cells, which are widely used as a model system for sympathetic ganglion-like neurons, were subjected to chemical hypoxia induced with rotenone, an inhibitor of mitochondrial complex I. Adenosine and its relatives guanosine and inosine were tested for their neuroprotective capability to improve neurite outgrowth and viability. In addition, cell lysates were analyzed for mitogen-activated-protein-kinases (MAPK) activation by anti-active and anti-total MAPKinase immunoblotting. Adenosine, guanosine and inosine significantly inhibited the loss of viability after hypoxic insult. In combination with NGF, purine nucleosides also partially rescued neurite outgrowth. The MEK-1/-2 inhibitor PD098059 inhibited purine nucleoside-mediated protection up to 85.23% and also markedly decreased neurite formation induced by NGF and purine nucleosides in hypoxic cells. Immunoblot analysis revealed a strong activation of MAPKinase upon incubation of cells with adenosine, guanosine or inosine. In combination with NGF an additive effect was observed. Results suggested that activation of the MAPKinase pathway plays a vital role in purine nucleoside-mediated protection of neuronal cells following hypoxic insult.     

5''-Nucleotidase activity and adenosine formation in stimulated, hypoxic and underperfused rat heart.

Changes in 5'-nucleotidase activity were calculated on the basis of alterations in ATP, ADP, phosphocreatine, Pi, Mg2+, IMP and AMP, determined by using 31P n.m.r. spectroscopy and h.p.l.c., during isoprenaline infusion, graded hypoxia and graded underperfusion in isolated rat heart. Calculated activity changes were compared with the total efflux of purines (adenosine + inosine + hypoxanthine) in order to assess the involvement of various 5'-nucleotidases in formation of adenosine. Purine efflux exhibited an exponential relation with cytosolic [AMP] during isoprenaline infusion and hypoxia (r = 0.92 and 0.95 respectively), supporting allosteric activation of 5'-nucleotidase under these conditions. Purine efflux displayed a linear relation with cytosolic [AMP] during graded ischaemia (r = 0.96), supporting substrate regulation in the ischaemic heart. The calculated activities of membrane-bound ecto-5'-nucleotidase were similar to the observed relations between purine efflux and cytosolic [AMP] in all hearts. The calculated activities of the ATP-activated cytosolic and lysosomal enzymes and of the ATP-inhibited cytosolic 5'-nucleotidase could not explain the observed release of purines under the conditions examined. These results indicate that the kinetic characteristics of the membrane-bound ecto-enzyme are consistent with an important role in the formation of extracellular adenosine, whereas the characteristics of the other 5'-nucleotidases are inconsistent with roles in adenosine formation under the conditions of the present study.     

A purine-selective nucleobase/nucleoside transporter in PK15NTD cells

Nucleoside and nucleobase transporters are important for salvage of purines and pyrimidines and for transport of their analog drugs into cells. However, the pathways for nucleobase translocation in mammalian cells are not well characterized. We identified an Na-independent purine-selective nucleobase/nucleoside transport system in the nucleoside transporter-deficient PK15NTD cells. This transport system has 1,000-fold higher affinity for nucleobases than nucleosides with K(m) values of 2.5 +/- 0.7 microM for [(3)H]adenine, 6.4 +/- 0.5 microM for [(3)H]guanine, 1.1 +/- 0.1 mM for [(3)H]guanosine, and 4.2 +/- 0.5 mM [(3)H]adenosine. The uptake of [(3)H]guanine (0.05 microM) was inhibited by other nucleobases and nucleobase analog drugs (at 0.5-1 mM in the order of potency): 6-mercaptopurine = thioguanine = guanine > adenine > thymine = fluorouracil = uracil. Cytosine and methylcytosine had no effect. Nucleoside analog drugs with modification at 2' and/or 5 positions (all at 1 mM) were more potent than adenosine in competing the uptake of [(3)H]guanine: 2-chloro-2'-deoxyadenosine > 2-chloroadenosine > 2'3'-dideoxyadenosine = 2'-deoxyadenosine > 5-deoxyadenosine > adenosine. 2-Chloro-2'-deoxyadenosine and 2-chloroadenosine inhibited [(3)H]guanine uptake with IC(50) values of 68 +/- 5 and 99 +/- 10 microM, respectively. The nucleobase/nucleoside transporter was resistant to nitrobenzylthioinosine {6-[(4-nitrobenzyl) thiol]-9-beta-D-ribofuranosylpurine}, dipyridamole, and dilazep, but was inhibited by papaverine, the organic cation transporter inhibitor decynium-22 (IC(50) of approximately 1 microM), and by acidic pH (pH = 5.5). In conclusion, we have identified a mammalian purine-selective nucleobase/nucleoside transporter with high affinity for purine nucleobases. This transporter is potentially important for transporting naturally occurring purines and purine analog drugs into cells.