Ontogeny of adenosine 3',5'-monophosphate metabolism in guinea pig cerebral cortex. II. Development of responses to L-glutamate in the presence of adenosine or histamine.

The effects of L-glutamate and other dicarboxylic amino acids on the accumulation of adenosine 3',5'-monophosphate (cyclic AMP) in slices of cerebral cortex from strain 2 guinea pigs were examined using tissue from animals at 39 days gestation to 7 days after birth. Responses to glutamate were inhibited completely by adenosine deaminase or theophylline unless histamine was present. When tested in the presence of adenosine, glutamate increased cyclic AMP accumulation up to 10-fold at 39 days gestation; the response was maximal at 52 days gestation, and both the efficacy and potency of glutamate declined thereafter. While the effects of glutamate were smaller in the presence of histamine plus theophylline, the developmental pattern was similar to that in the presence of adenosine. The relative potencies of D-aspartate, kainate, and alpha-methyl-DL-glutamate were much greater in fetal than in adult tissue. Glutamic acid diethyl ester, N-acetyl glutamate or 2,3-diaminopropionate had no effect in fetal tissue either in the presence or absence of glutamate. Responses to glutamate in adult tissue were much more dependent upon the presence of calcium ions than were those in fetal tissue. It was concluded that responses to glutamate involve mechanisms that differ in fetal and adult tissue.

Evidence for cross-linking of cyclic AMP to constituents of brain tissue by aldehyde fixatives: potential utility in histochemical procedures.

The amount of cyclic AMP recovered from unfixed tissue sections of brain carried through immunohistochemical procedures was found to be small (less than 2 pmoles/mg protein) and constant despite wide variations in the amount accumulated prior to freezing and sectioning. However, treatment of brain slices with glutaraldehyde or formaldehyde in buffered sucrose solutions for one to two hours at 0 degrees rendered insoluble as much as 60% of the total accumulated cyclic AMP as judged by filtration of homogenates of treated tissue. Immunoreactive cyclic AMP was recovered from filters by extraction with warm, dilute acid. The fraction of filter-bound cyclic AMP was relatively constant over a wide range of initial tissue levels. The persistence of insoluble cyclic AMP was greatest in homogenates of slices treated with formaldehyde when maintained above pH 8.5; about 40% of this fraction was lost after two hours at 0 degrees. Incubation of tissue homogenates with formaldehyde and radioactive nucleoside or nucleotide derivatives of adenine, guanine, and cytosine resulted in a time-dependent appearance of insoluble radioactivity; compounds lacking an amino function were inactive. Pure proteins, including polylysine, also reacted with formaldehyde and 3H-cyclic AMP to produce radioactivity resistant to adsorption by charcoal. The reaction between cyclic AMP or adenosine, formaldehyde, and alkyl amines was examined using UV spectrometry. It is tentatively concluded that at 0 degrees formaldehyde is capable of rapidly producing a methylene-bridged adduct between primary alkylamines and the N6-amino function of purine nucleosides or nucleotides. Application of these results to the development of immunohistochemical procedures for the cellular localization of cyclic AMP will require the generation of antibody preparations with high reactivity for cyclic AMP derivatized at the N6-position.