METABOLIC STUDIES OF GUINEA-PIG BASOPHILIC LEUKOCYTES IN SHORT-TERM TISSUE-CULTURE .1. MEASUREMENT OF HISTAMINE-SYNTHESIZING CAPACITY BY USING AN ISOTOPIC THIN LAYER CHROMATOGRAPHIC ASSAY

  • 1 January 1976
    • journal article
    • research article
    • Vol. 117  (4) , 1085-1092
Abstract
Mature circulating guinea pig basophils, purified to comprise 25% or more of leukocytes, were successfully maintained in short-term tissue culture for up to 72 h. These cells retained the ability to synthesize histamine, as assayed by a new isotopic-thin layer chromatographic assay which can reliably detect as little as 0.5 pg of 3H-histamine. Cell-associated, newly synthesized histamine was detectable as early as 1 h of culture, was substantially increased at 6 h, and reached maximal levels at 24 h, when it accounted for approximately 6.5% of total cell histamine. Newly synthesized histamine was still detectable at 48 and 72 h of culture. Histamine synthesis was decreased by lowering the concentration of histidine in the culture medium, and was markedly reduced by all of the specific histidine decarboxylase (HDC) inhibitors tested, but not by .alpha.-methyl-DOPA, pyrilamine maleate or metiamide. Increasing the concentration of pyridoxal phosphate, the HDC coenzyme, above that normally present in culture medium resulted in only an equivocal increase in the amount of newly synthesized histamine. Aminoguanidine, an inhibitor of histaminase, had no detectable effect. Uptake of exogenous histamine by cultured basophils was trivial compared to histamine synthesis. Both newly synthesized and previously manufactured, nonisotopic, histamine seemed to be stored in the same pool, as the same proportion of both was released by concanavalin A (Con A). Cellular histamine was largely conserved, with little or no spontaneous release into the medium of detectable isotopic or nonisotopic histamine. These techniques provide a model for studying granulocyte metabolic processes in vitro.