A simple and sensitive gas-chromatographic method for the determination of N-acetyl-L-aspartic acid (NA-Asp), N-acetyl-alpha-aspartylglutamic acid (NA-Asp-Glu) and beta-citryl-L-glutamic acid (beta-CG) was developed. The organ, regional and phylogenetic distributions of these compounds were studied. NA-Asp and NA-Asp-Glu were highly concentrated in nervous tissue, and less than 1% of the amounts in the nervous tissues were found in non-nervous organs. These two compounds showed a reciprocal relationship in their regional distribution in mature brains, but such a relationship was not evident or was even reversed in immature brains. The two compounds also showed different developmental changes in different regions of the brain. Fish brain contained a relatively high concentration of NA-Asp, but only a trace amount of NA-Asp-Glu. By contrast, a 10 times higher concentration of NA-Asp-Glu than NA-Asp was found in frog brain. Reptilian brain contained similar amounts of each compound. Avian and mammalian brain had NA-Asp at a roughly 10 times higher concentration than NA-Asp-Glu. beta-CG occurred at the highest concentration in the immature brain of rat and guinea pig, but disappeared in the mature brains. The adult frog brain, however, contained a large amount of beta-CG. In the adult rat, testis contained the highest concentration of beta-CG.

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The cat adrenal gland was retrogradely perfused with a medium lacking divalent cation, and the secretion of catecholamines was induced by Ca reintroduction with or without simultaneous removal of external Na. These responses were markedly reduced after prior exposure to a medium containing Mg (1 mM) or lacking Na. The inhibition caused by Mg was reversed by ouabain treatment. Reversing the concentration gradient for Na alone by replacing external Na with Tris, choline, or Li did not stimulate catecholamine secretion when Ca2+ (0.1 mM) was present in the external medium throughout the experiment. However, the treatment with ouabain reversed this inhibitory effect of Ca so that Na removal did induce marked secretion. Essentially similar results concerning secretion were obtained in the cultured bovine chromaffin cells. There was a reasonable correlation between secretion and 45Ca uptake in these cells under various experimental manipulations, and alterations of these parameters were well correlated with the level of internal Na. Furthermore, the rate constant of 22Na efflux was found to increase when Ca was reintroduced with the simultaneous removal of Na during exposure to a medium containing ouabain. These results are consistent with the view that the mechanism of internal Na-dependent Ca influx is activated to induce catecholamine secretion whenever internal Na is raised above a critical level. On the other hand, there were significant increases in the catecholamine secretion and 45Ca uptake which were induced by substitution of NaCI with sucrose, even when the operation of the internal Nadependent Ca influx mechanism was markedly restricted by various experimental manipulations. This suggests that other secretory mechanisms are involved under these conditions. This would account for the largest secretory effect of sucrose substitution under the condition in which internal Na-dependent Ca influx is activated.