Adenosine Inhibition of 45Ca Uptake by Synaptosomes as Function of Time of Electrical Stimulation

Abstract

The effect of adenosine on ⁴⁵Ca uptake by rat brain synaptosomes electrically stimulated was studied as function of time of stimulation (10, 30, 120 s). Inhibition of ⁴⁵Ca uptake was more evident for 120 s.     

Inhibition of Glycolysis by Adenosine Triphosphate

The effect of Adenosine triphosphate in inhibiting glycolysisin mitochondrial extracts from pea cotyledons was studied. Itwas possible to show a direct inhibition of adenosine triphosphateon glyceraldehyde phosphate dehydrogenase and on alcohol dehydrogenase.This inhibition was sufficient to explain the inhibition ofglycolytic phosphorlation observed previously.     

Uptake of Adenosine by Isolated Rat Brain Capillaries

Abstract: Adenosine uptake by isolated rat brain capillaries is a carrier-mediated, temperature- and pH-sensitive process. The K _m value for adenosine uptake is 4.74 μ m and the V _max is 21.7 picomol/mg protein/10 min. This is a high-affinity uptake system that can be cross-inhibited by several nucleosides and by the adenosine analogs tubercidin and 5'-deoxyadenosine. The uptake is very sensitive to inhibition by papaverine, hexobendine, and dipyridamole. These results confirm the existence of a nucleoside transport system associated with the blood-brain barrier observed during in vivo studies.     

Differentiating Adenosine Receptors and Adenosine Uptake Sites in Brain

Abstract

The characteristics of adenosine receptors and adenosine uptake sites in brain are presented. High affinity adenosine receptors of the A₁ type bind [³H]cyclohexyladenosine ([³H]CHA) and [³ H]diethyl-phenyl-xanthine ([³H]DPX) with 10^?9 potency while adenosine uptake sites are labeled 10^?10 potency with [³ H]nitrobenzyl-thioinosine ([³H]NBI). NBI does not inhibit either [³H]CHA (agonist) or [³H]DPX (antagonist) binding to adenosine receptors in brain cortical membranes and conversely CHA and other adenosine receptor ligands are very poor inhibitors of [³H]NBI binding to adenosine uptake sites. A number of other differences between the receptor and uptake site are discussed which provide rather strong evidence that these two sites are quite distinct and that the labeled ligands used represent specific probes for each site.     

Adenosine Triphosphate-Dependent Calcium Uptake by Rat Submaxillary Gland Microsomes

Microsomes from rat submaxillary glands are able to take up calcium from the suspension media. Calcium uptake is greatly increased by the presence of ATP. This effect of ATP is not detected at 0°C. ADP cannot replace ATP to potentiate calcium uptake. ATP-dependent calcium uptake is not observed in the absence of magnesium. ATP-dependent calcium uptake is enhanced by oxalate and, to a lesser degree, by inorganic phosphate. Total calcium per milligram of microsomal protein observed when tests were performed without oxalate closely parallels the amounts for skeletal and cardiac muscles reported by several authors. Calcium uptake in salivary gland microsomes is slower than in muscle microsomes. Speculations are considered about the role of ATP-dependent calcium uptake. It is suggested that a decrease in intracellular free calcium levels returns these cells to the resting state after secretion.     

Adenosine Inhibition of Human Platelet Aggregation by ADP

WE have shown that in human platelet-rich plasma, inosine is a concentration-dependent inhibitor of adenosine incorporation into platelets^1,2 and at high concentrations inosine inhibits adenosine decomposition³. This prompted us to investigate the effect of inosine and other adenosine decomposition products on aggregation of human platelets in vivo by ADP.     

Uptake of Adenosine by Cultured Cerebral Vascular Smooth Muscle Cells

Abstract: Adenosine uptake by cerebral smooth muscle cells is a carrier-mediated process. The K_m value for adenosine uptake is 10.0 μ M and the V _max is 0.95 nmol/ min-mg cell protein. This uptake system is inhibited by the adenosine analog 2-chloroadenosine at low adenosine concentrations. These results prove the existence of a nucleoside transport system associated with cerebral smooth muscle.     

Inhibition of Synaptosomal Neurotransmitter Uptake by Hallucinogens

The effect of 13 hallucinogens on the uptake of serotonin and norepinephrine into hippocampal synaptosomes and of serotonin and dopamine into caudate synaptosomes was found to be inhibitory, except for lysergic acid diethylamide and 2-bromolysergic acid diethylamide, which were inactive. The indoleal-kylamines were generally more potent than the phenylethylamines. The reported inhibition of uptake of serotonin by 5-methoxy-N,N-dimethyltryptamine and lysergic acid diethylamide into whole brain synaptosomes was not reproducible at concentrations 10² to 10⁴ times higher than those stated in the literature.     

Inhibition by Aspirin of Release of Antiheparin Activity from Human Platelets

Both in vitro and in vivo, aspirin inhibited the adenosine diphosphate and collagen-induced release of platelet factor 4 (antiheparin factor). The release induced by adrenaline and thrombin was not affected. The in-vivo effect in normal persons lasted for at least three days. Platelet uptake of acetyl-¹⁴C-aspirin was significantly greater than that of carboxyl-¹⁴C-aspirin.     

Solubilization of an Adenosine Uptake Site in Brain

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.     

Adenosine Triphosphate Inhibition of Yeast Trehalase

Yeast trehalase has been found to be inhibited non-competitively by adenosine triphosphate. Such a biological control could explain the accumulation of trehalose during the stationary phase of the growth curve.     

Radiation Inhibition of Amino Acid Uptake by Escherichia coli

The inhibition of macromolecular synthesis in Escherichia coli by ionizing radiation has been investigated. The survival of the ability to incorporate arginine, leucine, isoleucine, histidine, uracil, and glucose after various doses of gamma radiation, deuteron and alpha particle bombardment has been measured. All amino acids are incorporated by processes which show the same radiation sensitivity. The sensitivity of uracil corresponds to a volume which is roughly spherical, of radius about 160A, whereas the amino acids possess sensitive regions which are long and thin in character. The uptake of glucose is concerned with a smaller, roughly spherical unit. The possible identification of the radiation-sensitive targets with cellular constituents is discussed. The long thin character observed for amino acids suggests that the sensitive region affected by radiation is an unfolded form of a ribosome, or alternatively a long nucleic acid molecule. For uracil the sensitive region fits with a 70S ribosome, while for glucose a smaller particle would fit the data.     

Noncompetitive Amine Uptake Inhibition by the New Antidepressant Pridefine

Abstract: Pridefine (AHR-1118) is a pyrrolidine derivative with clinically established antidepressant efficacy. Previous work from this laboratory indicates that pridefine is a reuptake blocker of catecholamines and serotonin with weak releasing activity. This study characterized the mode of amine uptake inhibition by pridefine as noncompetitive. The uptake experiments were performed utilizing ouabain instead of zero-degree controls to differentiate between the passive and active components of uptake. Furthermore, the passive component was resolved into diffusion and binding of substrate. Correction was made for the effects of ouabain on binding. Kinetic constants determined from Lineweaver-Burk plots were: K_m= 3 × 10^?7 M for NE, K_m= 9 × 10^?8 M for DA, and K_m= 3 × 10^?8 M for 5-HT. Dixon analyses of uptake at various pridefine concentrations indicated noncompetitive inhibition with K_i= 2.5 × 10^?6 M for NE uptake, K_i= 2.0 × 10^?6 M for DA uptake, and K_i= 1 × 10^?5 M for 5-HT uptake. These constants compare well with IC₅₀ values for the same transmitters: NE, IC₅₀= 2.4 × 10^?6 M; DA, IC₅₀= 2.8 × 10^?6 M; 5-HT, IC₅₀= 1.0 × 10^?5 M. The in vitro results indicate that pridefine is relatively specific as a catecholamine uptake blocker. It differs from tricyclic antidepressants which are reportedly competitive inhibitors of monoamine uptake. The possible mechanisms by which pridefine acts as a noncompetitive inhibitor are discussed.     

Adenosine and Cyclic AMP in Rat Cerebral Cortical Slices: Effects of Adenosine Uptake Inhibitors and Adenosine Deaminase Inhibitors

The endogenous levels of adenosine functionally linked to cyclic AMP systems in rat cerebral cortical slices are regulated by both adenosine deaminase and adenosine uptake systems. 2'-Deoxycoformycin (2'-DCF), an adenosine deaminase inhibitor, slightly increased basal, adenosine, and norepinephrine-elicited accumulations of cyclic AMP, whereas dipyridamole, an uptake inhibitor, had an even greater effect on cyclic AMP accumulations under the same conditions. Combinations of 2'-DCF and dipyridamole elicited a greater effect than either compound alone. Neither 2'-DCF nor dipyridamole significantly augmented accumulations of cyclic AP elicited by a depolarizing agent, veratridine, suggesting that the adenosine "released" during neuronal depolarization of brain slices is not as subject to inactivation by uptake or deamination as endogenous adenosine in control brain slices. The accumulation of cyclic AMP elicited by a combination of norepinephrine and veratridine was greater than additive. The response to a pure beta-adrenergic agonist, isoproterenol, was not potentiated by 2'-DCF, dipyridamole, or veratridine, consonant with minimal interaction of endogenous adenosine with beta-adrenergic systems.     

Inhibition of Fumonisin B1 Cytotoxicity by Nanosilicate Platelets during Mouse Embryo Development

Nanosilicate platelets (NSP), the form of natural silicate clay that was exfoliated from montmorillonite (MMT), is widely used as a feed additive for its high non-specific binding capacity with mycotoxins such as fumonisin B₁ (FB₁), and has been evaluated its safety for biomedical use including cytotoxicity, genotoxicity, and lethal dosage (LD). In the study, we further examined its toxicity on the development of CD1 mouse embryos and its capacity to prevent teratogenesis-induced by FB₁. In vitro cultures, NSP did not disturb the development and the quality of intact pre-implantation mouse embryos. Further, newborn mice from females consumed with NSP showed no abnormalities. NSP had an unexpected high adsorption capacity in vitro. In contrast to female mice consumed with FB₁ only, a very low residual level of FB₁ in the circulation, reduced incidence of neutral tube defects and significantly increased fetal weight were observed in the females consumed with FB₁ and NSP, suggesting a high alleviation effect of NSP on FB₁in vivo. Furthermore, FB₁ treatment disturbed the gene expression of sphingolipid metabolism enzymes (longevity assurance homolog 5, LASS 5; sphingosine kinase 1, Sphk1; sphingosine kinase 2, Sphk2; sphingosine 1- phosphate lyase, Sgpl1; sphingosine 1-phosphate phosphatase, Sgpp1) in the maternal liver, uterus, fetus, and placenta, but NSP administration reversed the perturbations. Based on these findings, we conclude that NSP is a feasible and effective agent for supplementary use in reducing the toxicity of FB₁ to animals.     

Inhibition by Methylmalonate of Glycine Uptake by Synaptosomes from Rat Spinal Cord

Methylmalonate is accumulated in the genetically linked deficiency of methylmalonyl-CoA mutase (methylmalonic acidemia). In this condition is also observed an elevation of the glycine levels. This communication reports the inhibition of the synaptosomal glycine uptake by methylmalonate, when present at similar concentrations to those found in methylmalonic acidemia. This inhibition could be responsible, at least in part, for the neurological damage characteristic of this disease, by increasing the glycine levels in the synaptic cleft and thus interfering with the normal function of the inhibitory glycinergic synapsis in the spinal cord.     

Inhibition of Adenosine Triphosphatase in Sheep Red Cell Membranes by Oxidized Glutathione

This paper reports inhibition of Na⁺ + K⁺-stimulated, ouabain-inhibited adenosine triphosphatase (S-ATPase) in sheep red cell membranes by oxidized glutathione (GSSG). The results are consistent with the hypothesis that this inhibition depends upon the formation of a mixed disulfide between glutathione and -SH group(s) in the enzyme protein. Thus, inhibition of S-ATPase by GSSG proceeds more rapidly at alkaline than at neutral pH and is reversed by the addition of an excess of a compound containing reduced -SH groups (e.g. dithiothreitol). ATP protects S-ATPase against inhibition by GSSG and this protection depends on both the monovalent and divalent cation composition of the medium. Protection by ATP is more complete in the presence of K⁺ than in the presence of Na⁺.     

Identification of the Adenosine Uptake Sites in Guinea Pig Brain

Nitrobenzylthioinosine (NBMPR), a potent and specific inhibitor of nucleoside transport, was employed as a photolabile probe of the adenosine transporter in guinea pig brain membranes. Reversible, high-affinity binding of [3H]NBMPR to a crude preparation of guinea pig brain membranes was demonstrated (apparent KD 0.075 +/- 0.012 nM; Bmax values of 0.24 +/- 0.04 pmol/mg protein). Adenosine, uridine, dipyridamole, and nitrobenzylthioguanosine inhibited high-affinity binding. Low concentrations of cyclohexoadenosine (10-300 nM) had no effect on NBMPR binding. These properties of the high-affinity NBMPR binding sites were consistent with NBMPR binding to the nucleoside transport protein. Exposure of brain membranes in the presence of [3H]NBMPR and dithiothreitol, a free-radical scavenger, to ultraviolet light resulted in covalent incorporation of 3H into polypeptides of apparent MW 66,000-45,000, a value similar to that for the human erythrocyte nucleoside transporter. Covalent attachment of [3H]NBMPR was inhibited by adenosine, dipyridamole, and nitrobenzylthioguanosine.     

Glutamate Uptake into Synaptic Vesicles: Competitive Inhibition by Bromocriptine

The ATP-dependent uptake of L-glutamate into synaptic vesicles has been well characterized, implicating a key role for synaptic vesicles in glutamatergic neurotransmission. In the present study, we provide evidence that vesicular glutamate uptake is selectively inhibited by the peptide-containing halogenated ergot bromocriptine. It is the most potent inhibitor of the agents tested: the IC50 was determined to be 22 microM. The uptake was also inhibited by other ergopeptines such as ergotamine and ergocristine, but with less potency. Ergots devoid of the peptide moiety, however, such as ergonovine, lergotrile, and methysergide, had little or no effect. Although bromocriptine is known to elicit dopaminergic and serotonergic effects, its inhibitory effect on vesicular glutamate uptake was not mimicked by agents known to interact with dopamine and serotonin receptors. Kinetic data suggest that bromocriptine competes with glutamate for the glutamate binding site on the glutamate translocator. It is proposed that this inhibitor could be useful as a prototype probe in identifying and characterizing the vesicular glutamate translocator, as well as in developing a more specific inhibitor of the transport system.