Worm Breeder's Gazette 12(5): 67 (February 1, 1993)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

5-Ht (Serotonin) Metabolism: Identification of a Developmentally Regulated 5-Ht N-Acetyltransferase

Richmond Muimo, R. Elwyn Isaac

Department of Pure and Applied Biology, University of Leeds, Leeds LS2 9JT, U.K.

5-HT is found in a number of neurons in C elegans including the Hermaphrodite Specific Neurons (HSN) which innervate the sex muscles of hermaphrodites and control egg-laying (Desai et al., 1988, Nature 336, 638-646). The addition of 5-HT to worms in M9 results in stimulation of egg-laying and mutants (e.g.. egl-l, egl-5 and egl-10 )with HSN abnormalities become bloated with eggs but respond to exogenously applied 5-HT by laying eggs, demonstrating an important regulatory role for 5-HT (Trent et al.,1983, Genetics 104, 619-647).

We are interested in the inactivation of biogenic amines in nematodes and have studied the metabolism of 5-HT in C. elegans. Initially, we incubated cut worms with [14C]5-HT and after 2h at 20°C, the radiolabelled metabolites were extracted and analyzed .together with marker compounds on two dimensional silica TLC. Autoradiograms of the developed plates identified the major metabolite as N-acetyl 5-HT and the identity was confirmed by chromatography on two different reversed phase HPLC systems. There were no metabolites corresponding to products of oxidative deamination. N-acetylation of [14C]5-HT by C. elegans homogenate was dependent upon the addition of acetyl CoA to the incubation mixture. The N-acetyltransferase (NAT) was localized in a cytosol fraction and displayed Michaelis-Menten kinetics with a high affinity for 5HT (apparent Km, 30 +/- 8 mM; +/- SEM, n=3). Soluble enzyme was prepared from staged worms (L1, L2 , L3 , L4 and adults) and the specific activity of the NAT in each preparation determined from linear reaction rates (fig.). The specific activity was very low (< 8 nmol NA-5HT/h/mg of protein) in L1 and L2 stages but increased in an exponential manner with development, reaching the highest activity ( 144 nmol NA-5HT/h/mg of protein) in adult worms.

[See Figure]

The increase in the specific activity of the 5-HT NAT occurring at around the L4 /adultstage correlates with the appearance of 5-HT in the HSN cells and the appearance of the sex muscles that are innervated by these neurons. This correlation and the high affinity for 5-HT is consistent with a physiological role for the NAT in the metabolism of this amine but it is also possible that other biogenic amines, such as dopamine and octopamine, can also serve as substrates for this enzyme. We are also considering the possibility that N-acetylated amines are not necessarily end-products of metabolism but may have a signaling role in their own right. Future work will focus on defining the precise function of this enzyme in amine metabolism in adult nematodes.