Alexander S. Bender

The Characterization of [3H] Adenosine Uptake into Rat Cerebral Cortical Synaptosomes

: Uptake of adenosine, a putative inhibitory transmitter or modulator, was investigated in rat cerebral cortical synaptosomes. The accumulation of [3H]adenosine into synaptosomes, using an adenosine concentration of 10 μ.m, was linear for 30 min at 37°C. The uptake appeared to be mediated by kinetically saturable processes with apparent Kms of 1 μam (“high‐affinity A”) and 5 μm (“high‐affinity B”), both of which were partially sensitive to the presence of external sodium and calcium ions. Both uptake processes were partially inhibited by 2,4‐dinitrophenol, implying the presence of active uptake and diffusional components. A study of the metabolites of adenosine taken up by the two uptake systems indicates that the major metabolites were adenosine and nucleotides. However, adenosine incorporated by the high‐affinity A uptake system is more likely to form deaminated metabolites, such as hypoxanthine and inosine, indicating a possible functional difference between the two uptake processes. A detailed comparison of the inhibitory properties of certain adenosine analogues and other pharmacological agents has revealed differences between the two adenosine uptake systems. Since the glial contamination in synaptosomal preparations is well established, one of the uptake systems we observed in the present study might be of glial origin. This notion is supported by the findings that the Km values and kinetic properties of papaverine action in the synaptosomal high‐affinity A uptake system are similar to those of astrocytes reported in the literature. In conclusion, the uptake processes of synaptosomal preparations show that accumulation of adenosine into neuronal (and possibly glial) elements may play a major role in regulating the extracellular adenosine concentration. Uptake inhibitors, such as diazepam, may exert, at least in part, their pharmacological actions by interfering with the regulation of extracellular adenosine concentrations.

The Rapid Uptake and Release of [3H]Adenosine by Rat Cerebral Cortical Synaptosomes

Abstract: Adenosine, a putative inhibitory transmitter or modulator in the brain, is rapidly transported by rat cerebral cortical synaptosomes. The uptake may represent a facilitated diffusion process, which is saturable and temperature‐dependent. In this study, the uptake process was very rapid, reaching completion within 60 s of incubation at 37°C, and had an apparent Km value of 0.9μM and a Vmax value of 5.26 pmol/mg protein/ 30 s. Over 70% of the adenosine taken up remained unchanged, whereas 14% was metabolized to inosine. Twelve percent of the adenosine was converted to nucleotides. Rapid uptake of adenosine into rat cerebral cortical synaptosomes was partially inhibited by replacing Na+ with choline chloride in the medium. Ca2+ ion is important for the uptake process, as inhibition of adenosine uptake occurs in the presence of either Co2‐ or EGTA. Rapid uptake of adenosine is apparently mediated by a nucleoside carrier, a conclusion based on its inhibition by a variety of purine and pyrimidine nucleosides. Uptake was inhibited by dipyridamole, hexobendine, papaverine, flurazepam, and morphine. Over 60% of the adenosine taken up by the rapid uptake system (30 s) was released by depolarizing agents. In contrast, only 30% of the adenosine taken up during a 15‐min incubation period was released under the same conditions. [3H]Adenosine was the predominant purine released in the presence or absence of depolarizing agents. The basal and KCl‐evoked release mechanisms were found to be at least partially Ca2+‐dependent, however, the release of adenosine by veratridine was increased in the presence of EGTA. This finding is in agreement with the reported Ca2+‐independent release of ATP from brain synaptosomes. The present findings suggest that there are at least two functional pools of adenosine in synaptosomes. Adenosine taken up by different uptake systems may be destined for different uses (metabolism or release) in the neuron.

Osmotic regulation of myo-inositol uptake in primary astrocyte cultures

Uptake ofmyo-inositol by astrocytes in hypertonic medium (440 mosm/kg H2O) was increased near 3-fold after incubation for 24 hours, which continued for 72 hours, as compared with the uptake by cells cultured in isotonic medium (38 nmoles/mg protein).myo-Inositol uptake by astrocytes cultured in hypotonic medium (180 mosm/kg H2O) for periods up to 72 hours was reduced by 74% to 8 to 10 nmoles/mg protein. Astrocytes incubated in either hypotonic or hypertonic medium for 24 hours and then placed in isotonic medium reversed the initial down- or up-regulation of uptake. Activation of chronic RVD and RVI correlates with regulation ofmyo-inositol uptake. A 30 to 40 mosm/kg H2O deviation from physiological osmolality can influencemyo-inositol homeostasis. The intracellular content ofmyo-inositol in astrocytes in isotonic medium was 25.6 ± 1.3 μg/mg protein (28 mM). This level ofmyo-inositol is sufficient for this compound to function as an osmoregulator in primary astrocytes and it is likely to contribute to the maintenance of brain volume.

Similarities of adenosine uptake systems in astrocytes and neurons in primary cultures

Uptake of extracellular adenosine was studied in primary cultures of astrocytes or neurons. Both cell types showed a high affinity uptake. TheKm values were not significantly different (6.5±3.75 μM in astrocytes and 6.1±1.86 μM in neurons), but the intensity of the uptake was higher in astrocytes than in neurons (Vmax values of 0.16±0.030 and 0.105±0.010 nmol×min−1×mg−1 protein, respectively). The temperature sensitivity was similar in the two cell types. Adenosine uptake inhibitors and benzodiazepines inhibited the adenosine uptake systems in both astrocytes and neurons with IC50 values in the high nanomolar or the micromolar range and the rank order of potency was similar in the two cell types. In both cell types the (−) isomers of two sets of benzodiazepine stereoisomers were more potent than the (+) isomers. Dixon analysis showed that dipyridamole, papaverine, hexobendine and chlordiazepoxide inhibited the adenosine uptake competitively and clonazepam noncompetitively in both cell types.

Inhibition of adenosine uptake into rat brain synaptosomes by the benzodiazepines

Abstract 1. 1. Thirteen benzodiazepines have been tested for their ability to inhibit the uptake of adenosine by rat brain synaptosomes. 2. 2. All of these benzodiazepines were able to inhibit adenosine uptake with IC 20 values ranging from 5 × 10 −9 M (clonazepam) to 2.0 × 10 −5 M (RO 11-6893). 3. 3. Clonazepam, nitrazepam, lorazepam, RO 11-6896, diazepam, flunitrazepam, medazepam, and flurazepam were the most potent compounds, having IC 20 values of less than 10 −6 M. Adenosine uptake would therefore be appreciably reduced at the levels achieved with therapeutic doses (circa 1 μM for diazepam). 4. 4. The d-steroisomer RO 11-6896 was approximately 200 times more active than the 1-isomer, RO 11-6893. 5. 5. The high IC 50 values (10 −5 −10 −3 M) indicate that dose response curves for the benzodiazepines are rather shallow. 6. 6. The results are consistent with the hypothesis that benzodiazepines exert some of their actions via an enhancement of the extracellular levels of adenosine.

Flunitrazepam Binding to Intact and Homogenized Astrocytes and Neurons in Primary Cultures

Abstract: [3H]Flunitrazepam binds to intact and homogenized mouse astrocytes and neurons in primary cultures. In intact cells, the binding is to a single, high‐affinity, saturable population of benzodiazepine binding sites with a KD of 7 nM and Bmax of 6,033 fmol/mg protein in astrocytic cells and a KD of 5 nM and Bmax of 924 fmol/mg protein in neurons. After homogenization, the Bmax values decrease drastically in both cell types, but most in astrocytes. The temperature and time dependency are different for the two cell types, with a faster association and dissociation in astrocytes than in neurons and a greater temperature sensitivity in the astrocytes. Moreover, flunitrazepam binding sites on neuronal and astrocytic cells have different pharmacological profiles. In intact astrocytic cells, Ro 5–4864 (K1= 4 nM) is the most potent displacing compound, followed by diazepam (K1= 6 nM) and clonazepam (K1= 600 nM). In intact neurons, the relative order of potency of these three compounds is different: diazepam (K1= 7 nM) is the most potent, followed by clonazepam (K1= 26 nM) and Ro 5–4864, which has little effect. After homogenization the potency of diazepam decreases. We conclude that both neuronal and astrocytic cells possess high‐affinity [3H]flunitrazepam binding sites. The pharmacological profile and kinetic characteristics differ between the two cell types and are further altered by homogenization.

Do benzodiazepines bind at adenosine uptake sites in CNS

Abstract Benzodiazepines inhibit adenosine uptake into rat cerebral cortical synaptosomes and their potency as inhibitors of adenosine uptake is closely correlated with therapeutic efficacy. Agents which possess “benzodiazepine like” activities such as CL218,872, zopiclone and fominoben and which displace benzodiazepine binding to brain cell membranes, are also inhibitors of adenosine uptake into brain synaptosomes. The IC50 values of all these compounds as inhibitors of adenosine uptake are in close agreement with the IC50 values obtained for the displacement of benzodiazepine binding to the brain receptors. Adenosine uptake inhibitors (dipyridamole, hexobendine, papaverine, 6-(2-hydroxy-5-nitrobenzyl)thioguanosine) which competitively inhibit adenosine uptake, presumably by blocking adenosine binding to its carrier-protein, are competitive inhibitors of diazepam binding to the brain membrane receptors. The finding of a pronounced correlation between inhibition of benzodiazepine binding and inhibition of adenosine uptake further supports the proposal that benzodiazepines may exert part of their pharmacological action through the inhibition of adenosine uptake.

Some Biochemical Properties of the Rapid Adenosine Uptake System in Rat Brain Synaptosomes

Abstract: The rapid uptake of adenosine into rat brain cortical synaptosomes is mediated by a facilitated diffusion process. The carrier mediated uptake is sensitive to pH and temperature. The average Q10 value for the system is approximately 1.77 and the necessary activation energy (Ea) is estimated to be 8870 cal/mol. These values are essentially in agreement with values reported for adenosine uptake carriers of other tissues. Substrate specificity of the uptake system in the CNS demonstrates that nucleotides do not interact with the carrier until they have been hydrolyzed to nucleosides. Structural modification of the purine moiety at the “2” position did not have a profound effect on the ability of the molecule to serve as a substrate for the uptake system. Competitive inhibition by sulfhydryl reagents, p‐chloromercuribenzoate, and N‐ethylmaleimide on adenosine uptake suggests a direct involvement of sulfhydryl group(s) in the uptake mechanism. Other purines and pyrimidines also inhibited adenosine uptake, suggesting that a variety of nucleosides can interact with a common carrier system.

Binding of [3H]Ro 5–4864 in primary cultures of astrocytes

Intact primary cultures of astrocytes display benzodiazepine receptors, which can be labeled with [3H]Ro 5-4864. Binding of [3H]Ro 5-4864 is specific, saturable and temperature dependent, being maximal at 0 degrees C. Scatchard analyses show a single population of high affinity binding sites with a Kd value of 6.7 nM and a Bmax value of congruent to 12,000 fmol/mg protein. The binding reaches equilibrium at congruent to 100 min, with k+1 of 0.0078 nM-1 X min-1 and is rapidly reversible with k-1 of 0.057 min-1. [3H]Ro 5-4864 binding is not modulated by GABA. Certain benzodiazepines (flunitrazepam, diazepam, Ro 7-3351) and dipyridamole displace this binding with IC50 values in the nanomolar range, whereas other benzodiazepines (alprazolam, clonazepam, chlordiazepoxide) as well as carbamazepine, phenytoin and phenobarbital have IC50 values in the micromolar range. These characteristics resemble those of Ro 5-4864 binding to brain membrane preparations reported by other authors and thus indicate that the cultured astrocytes are good models of their in vivo counterparts.

Benzodiazepines and β-adrenergic binding to primary cultures of astrocytes and neurons

Characteristics of primary cultures of neurons or of astrocytes are discussed which suggest that such cultures are appropriate models for their in vivo counterparts. Advantages or disadvantages of these cultures for studies of receptor binding are: Neuronal and astrocytic binding can be studied separately. Binding can be studied to intact cells or after homogenization. Apparent binding to intact cells may include unspecific retention. This seems to be a problem for beta-adrenergic ligands but not for benzodiazepines.