Tryptophan Hydroxylase Activity in Serotonin Producing Mast Cells

Abstract

The monoamine, serotonin, has long been known to function as a hormone, neurotransmitter, and local chemical mediator. Enterochromaffin cells of gastrointestinal mucosa are believed to be the predominant source in the periphery and, in rodents, mast cells are also a source. Tryptophan hydroxylase (TPH) is the rate limiting enzyme in the biosynthesis of this monoamine. TPH of peripheral origin and brain TPH are known to differ with respect to their various properties (Nakata and Fujisawa, 1982; Hasegawa et al., 1987; Kim et al., 1991). The phosphorylated brain enzyme is known to be activated in the presence of 14-3-3 protein (Yamauchi et al., 1981), however, little is known concerning regulation of the enzyme of peripheral origin such as from mast cells and gastrointestinal enterochromaffin cells. In vitro activation of peripheral TPH by anaerobic incubation with DTT and Fe²⁺ included in routine measurement of this enzyme since the enzyme is very labile and must be re-activated prior to the assay (Ichiyama et al., 1974). With regard to the mechanism of activation, accumulated data from in vitro studies suggest that this activation requires supplementation with ferrous iron. Hence “DTT-activation” of the enzyme could be termed “ferrous iron activation”. Some degree of uncertainty has remained regarding whether the iron concentration in the living cells is inadequate for TPH activation. The question arose as to whether the intracellular enzyme is actually supplied with sufficient ferrous iron or even whether “ferrous iron activation” really occurs. The aim of this work was to answer this question. Intracellular enzyme active was assessed under conditions in which the iron concentration was manipulated using permeable chelators. Mouse bone marrow derived mast cells (BMMC) in culture were generally used in this work as a source of natural mast cells.