AMINO ACID DISTRIBUTION AND INCORPORATION INTO PROTEINS IN ISOLATED, ELECTRICALLY-STIMULATED CEREBRAL TISSUES

—The uptake of radioactive amino acid by incubated cerebral cortex slices is found to be a first order process. Incorporation of the radioactive amino acid into tissue protein is from a precursor pool that has first equilibrated with the intracellular endogenous free amino acids. Ways of calculating the amino acid incorporation in molar quantities from the observed incorporation of radioactivity are discussed, and it is concluded that the specific radioactivity of the intracellular acid-soluble fraction is the best basis for such estimates. The in vitro incorporation of leucine into tissue protein is estimated to be approximately 1±2 mμnol/mg protein/h, and of valine 0±4 mμmol/mg protein/h. Addition of free amino acids to the media had little or no effect on the calculated rates of incorporation. On incubation for 1 h the total free valine in tissue and medium increased by 0±43 μmol/g and leucine increased by 0±55 μmol/g. Estimates of amino acid incorporation based on the specific radioactivity of the media amino acids can give misleading results if this considerable release of amino acids into the medium is not taken into account. Electrical stimulation of neocortical slices with a variety of types of pulses was either without effect or decreased incorporation into portein. The decrease could not be directly correlated with changes in tissue K⁺ nor with the utilization of ATP. Mild, local stimulation of the lateral olfactory tract of piriform cortex slices was without effect on tissue phosphocreatine, K⁺ or amino acid incorporation.     

Effects of low-chloride solutions on action potentials of sheep cadiac purkinje fibers

The rapid repolarization during phase 1 of the action potential of sheep cardiac purkinje fibers has been attributed to a time- and voltage-dependent chloride current. In part, this conclusion was based on experiments that showed a substantial slowing of phase 1 when larger, presumably impermeant, anions were substituted for chloride in tyrode’s solution. We have re- examined the electrical effects of low-chloride solutions. We recorded action potentials of sheep cardiac purkinje fibers in normal tyrode’s solution and in low-chloride solutions made by substituting sodium propionate, acetylglycinate, methylsulfate, or methanesulfonate for the NaCl of Tyrode’s solution. Total calcium was adjusted to keep calcium ion activity of test solutions equal to that of control solutions. Propionate gave qualitatively variable results in preliminary experiments; it was not tested further. Low-chloride solutions made with the other anions gave much more consistent results: phase 1 and the notch that often occurs between phases 1 and 2 were usually unaffected, and the action potential duration usually increased. The only apparent change in the resting potential was a transient 3-6 mV depolarization when low-chloride solution was first admitted to the chamber, and a symmetrical transient hyperpolarization when chloride was returned to normal. If a time- and voltage-dependent chloride current exists in sheep cardiac purkinje fibers, our results suggest that it plays little role in generating phase 1 of the action potential.     

The binding of [3H]adenosine to synaptosomal and other preparations from the mammalian brain.

1. A high-affinity adenosine-binding site with Kd(adenosine) 0.5-1.3 microM was demonstrated in particulate and synaptosomal fractions isolated from the cerebral cortex of guinea pig, rat and ox. 2. Binding of [3H]adenosine to this site was inhibited by theophylline and by 2-chloroadenosine, but not by four other adenosine analogues. 3. Endogenous adenosine, found to be present in some preparations at approx. 1 pmol/mg of protein, diminished the binding capacity of the preparations for [3H]adenosine. 4. Addition of the adenosine deaminase inhibitor erythro-9-[1-(1-hydroxyethyl)heptyl]-adenine revealed the presence of a second lower affinity binding site with Kd (adenosine) 5-9 microM and a higher maximal adenosine-binding capacity. The inhibitor partially blocked binding to the high-affinity site in preparations from which adenosine deaminase had been removed by washing. 5. To preparations of particulate fractions maintained under iso-osmotic conditions, adenosine attachment was non-saturable and temperature-dependent, indicating the existence of an active uptake process. 6. The location and binding constant of the high-affinity adenosine-binding site suggest that it corresponds to the receptor site for adenosine-activated adenylate cyclase.     

Adenine mononucleotides and their metabolites liberated from and applied to isolated tissues of the mammalian brain

Preincubation with [¹⁴C] adenine labeled the nucleotide fraction of isolated cerebral tissues, which subsequently released 0.18% of their¹⁴C content per minute, a proportion increased threefold by electrical excitation. Of the¹⁴C released, 2–3% was as 5-adenine nucleotides and about 2% as cyclic adenosine 35-monophosphate (cAMP). Among the 5-nucleotides AMP greatly preponderated, and ATP and ADP were detected. When added to (unlabeled) incubating neocortical tissue, ATP and AMP yielded adenosine as the major product, with smaller quantities of inosine and hypoxanthine, to effluent fluids. cAMP so added yielded 5-nucleotides and the other compounds named; adenosine yielded mainly inosine and hypoxanthine. Results from these reactions and others in which theophylline was included led to the conclusion that an appreciable proportion of the effluent [¹⁴C] adenosine, inosine, and hypoxanthine derived from cAMP.