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.     

Effect of 2-(4-Phenylpiperidino)cyclohexanol (AH 5183) on the Veratridine-Induced Increase in Acetylcholine Synthesis by Rat Hippocampal Tissue

The intent of this study was to determine whether the drug 2-(4-phenylpiperidino)cyclohexanol (AH 5183 or vesamicol) might inhibit the veratridine-induced increase in acetylcholine (ACh) synthesis by reducing the veratridine-induced activation of a detergent-soluble choline-O-acetyltransferase (EC 2.3.1.6; ChAT) fraction associated with a vesicle-bound store of ACh. When minces of rat hippocampal tissue were loaded with [14C]choline and subsequently depolarized with veratridine, an increase in the synthesis of [14C]ACh occurred that could be abolished by L-AH 5183 (75 nM). When minces were depolarized with veratridine in the presence of L-AH 5183 (75 nM), the depolarization-induced activation of a detergent-soluble ChAT fraction associated with a vesicle-bound store of ACh was blocked. Conversely, the veratridine-induced activation of a water-soluble ChAT fraction believed to be cytosolic was not. AH 5183 also blocked the repletion of the vesicle-bound store with newly synthesized ACh following veratridine-induced depletion of ACh, a result that appeared to be mediated by an effect on the synthesis of ACh at the vesicular surface. These results suggest that veratridine depolarization of rat hippocampal nerve terminals stimulates the synthesis of ACh by activating a detergent-soluble fraction of ChAT closely associated with synaptic vesicle release sites. ACh synthesis and transport at the vesicular surface may be influenced by a common AH 5183-sensitive regulatory protein.     

Effect of Taurine on Calcium Accumulation in Resting and Depolarised Insect Synaptosomes

The effect of taurine (2-aminoethanesulphonic acid) on 45Ca2+ accumulation in resting and depolarised synaptosomes obtained from the locust Schistocerca americana gregaria was studied. Taurine reduced 45Ca2+ accumulation in resting synaptosomes, and this effect was more pronounced when synaptosomes were depolarised with either high [K+] or veratridine. Veratridine-induced 45Ca2+ accumulation was not affected by either gamma-aminobutyric acid or leucine, but was reduced by both verapamil and tetrodotoxin.     

Acetylation of Synaptosomal Protein: Inhibition by Veratridine

Abstract: Incubation of synaptosomes with [³H]acetate results in rapid labeling of protein. Labeling is decreased in the presence of veratridine, and the effect of veratridine is blocked by tetrodotoxin. Most of the radioactivity can be removed by base or acid hydrolysis, and is probably incorporated as acetate; it is this fraction that is affected by the veratridine. The data suggest that veratridine stimulates deacetylation of synaptosomal protein. This raises the question whether acetylation-deacetylation is involved in membrane function.     

Stimulation of Na+-Dependent, Veratridine-Sensitive Synaptosomal Amino Acid Uptakes by Serum Albumin

Abstract: Bovine serum albumin (BSA) is shown to stimulate selectively the synaptosomal uptakes of those amino acids that are dependent on external Na⁺ and that are inhibited by veratridine. Thus, the stimulation can be seen in the case of aspartic acid, glutamic acid, glycine, proline, and γ-aminobutyric acid, but not with serine and threonine. Further, studies on the interaction of veratridine, valinomycin, and BSA on the uptake of proline suggest that the primary action of the albumin is to increase the influx of proline. Such an action could result as a consequence of stabilization of the Na⁺ gradient by increased endogenous levels of ATP. Intrasynaptosomal ATP was increased in the presence of BSA but significantly decreased by veratridine.     

Continuous Monitoring of Extracellular Glucose Concentrations in the Striatum of Freely Moving Rats with an Implanted Glucose Biosensor

Abstract: We have used a glucose oxidase-based sensor implanted in the striatum of freely moving rats to determine the concentration of extracellular glucose in two distinct ways. With a modification of the zero net flux method, in which different concentrations of glucose are infused through a dialysis probe glued to the biosensor, we calculated the concentration at which there was no change in glucose current by regression analysis; this gave a concentration of 0.351 ± 0.016 m M . Calculating the concentration from the basal current and the in vitro calibration of the biosensor was not significantly different from this. The basal extracellular glucose concentration determined by either method remained constant over a period of several days. Infusion of 50 µ M veratridine through the adjacent dialysis probe caused a steep decrease in glucose current as soon as the drug reached the brain in contrast to the delayed fall (7.5 min) seen with microdialysis in previous experiments from this laboratory. These results demonstrate that this biosensor provides a direct, real-time measure of the extracellular concentration of glucose.     

7Li Nuclear Magnetic Resonance Study for the Determination of Li+ Properties in Neuroblastoma SH-SY5Y Cells

Abstract: Lithium has been used clinically in the treatment of manic depression. However, its pharmacologic mode of action remains unclear. Characteristics of Li⁺ interactions in red blood cells (RBCs) have been identified. We investigated Li⁺ interactions on human neuroblastoma SH-SY5Y cells by developing a novel ⁷Li NMR method that provided a clear estimation of the intra- and extracellular amounts of Li⁺ in the presence of the shift reagent thulium-1,4,7,10-tetrazacyclododecane- N,N ', N ", N ‴-tetramethylene phosphonate (HTmDOTP⁴⁻). The first-order rate constants of Li⁺ influx and efflux for perfused, agarose-embedded SH-SY5Y cells in the presence of 3 m M HTmDOTP⁴⁻ were 0.055 ± 0.006 (n = 4) and −0.025 ± 0.006 min⁻¹ (n = 3), respectively. Significant increases in the rate constants of Li⁺ influx and efflux in the presence of 0.05 m M veratridine indicated the presence of Na⁺ channel-mediated Li⁺ transport in SH-SY5Y cells. ⁷Li NMR relaxation measurements showed that Li⁺ is immobilized more in human neuroblastoma SH-SY5Y cells than in human RBCs.     

Parallel but Separate Release of Catecholamines and Acetylcholinesterase from Stimulated Adrenal Chromaffin Cells in Culture

Abstract: A pharmacological study was made of the effects of veratridine and lasalocid on the release of catecholamines, acetylcholinesterase (AChE) and dopamine-β-hydroxylase (DBH) from cultures of isolated bovine adrenal chromaffin cells. Exposure of the cultures to veratridine resulted in concomitant release of catecholamines and AChE into the external medium in a dose-dependent and Ca^{2 +}-dependent manner. A Ca²⁺ iono-phore, lasalocid, also produced a dose-dependent and parallel release of both catecholamines and AChE. The release of the two components was accompanied by release of DBH. The present results provide pharmacological evidence for a parallel release of catecholamines, AChE, and DBH from cultured adrenal chromaffin cells, and the stoichiometry of the release evoked by different secretagogues suggests that AChE and catecholamines are released from different cellular compartments.     

Insulin Is Released from Rat Brain Neuronal Cells in Culture

Depolarization of neuronal cells in primary culture from the rat brain by potassium ions in the presence of calcium or by veratridine caused a greater than three-fold stimulation of release of immunoreactive insulin. HPLC of the released insulin immunoreactivity from the neuronal cultures comigrated with the two rat insulins. The depolarization-induced release of insulin was inhibited by cycloheximide and was specific for neuronal cultures since potassium ions failed to cause the release in comparably prepared astrocytic glial cells from the rat brain. Prelabelling of neuronal cultures with [3H]leucine followed by depolarization resulted in the release of radioactivity that immunoprecipitated with insulin antibody. The release of [3H]insulin was biphasic. These observations suggest that neuronal cells from the brain have the capacity to synthesize insulin that could be released under depolarization conditions.     

Glycine release from Y79 retinoblastoma cells

Abstract: Glycine release, induced by a high concentration of potassium chloride (K⁺), was investigated in cultured human Y79 retinoblastoma cells. The cells were labeled by incubation with [2-³H]glycine prior to K⁺ depolarization. Depolarization with 55 m M K⁺ caused an immediate, Ca²⁺-dependent release of approximately 20% of the cellular radiolabeled glycine content. Chemical analysis of the intracellular free glycine content also showed that approximately 20%, 2.4 nmol/mg protein, was released after K⁺ depolarization. Glycine release from labeled Y79 cells was not stimulated by incubation with 55 mM choline chloride. Based on measurements with an amino acid analyzer, it is concluded that of the free amino acids contained in the Y79 cell, only glycine is specifically released into the extracellular fluid by K⁺ depolarization. Although the intracellular content of serine and glutamate decreased, these amino acids were not released from the cells. Further studies with [U-¹⁴C]serine suggest that serine is converted into glycine in Y79 cells. Veratridine also caused an immediate release of [2-³H]glycine from the cells, and this was blocked by tetrodotoxin. This suggests that the Y79 cells possess voltage-dependent Na⁺ channels. These results indicate that K ⁺ - and veratridine-stimulated glycine release occurs in Y79 retinoblastoma cells, providing additional evidence that this continuously cultured line may be a useful model for certain human retinal and central nervous system functions.