Worm Breeder's Gazette 12(3): 52 (June 15, 1992)
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.
Monoamine oxidase A and B are mitochondrial outer membrane flavoproteins best characterized in higher vertebrates, and their association with the nervous system has led to the belief that their most important function is a role in neurotransmitter amine metabolism. In spite of extensive efforts to understand the role of these enzymes in the nervous system, little progress has been made. This is in part due to the inherent complexity of the systems studied. Because the nervous system of Caenorhabditis elegans is relatively simple and well defined, I decided to investigate if MAO is present in C elegans. The biogenic amines octopamine, serotonin, and dopamine, presumptive neurotransmitters in the worm, are all substrates of MAO. It is therefore reasonable to believe that MAO is present in C. elegans. Although first experiments to spectrophotometrically detect MAO activity failed, immunological studies of whole worm extracts indicated its presence. Homogenates were examined by SDS-PAGE and Western blot analysis with sheep anti-human MAO A, rabbit anti-human MAO A and sheep anti-bovine MAO B antibodies. A strong and specific alkaline phosphatase reaction was obtained with both anti-human MAO A antibodies. No trace of a signal was detected with anti-bovine MAO B antibody or preimmune sera. The positive band migrated more slowly than the human control suggesting that the putative nematode MAO is about 5 to 7kDa larger than the human enzyme.
Evidence for the presence of MAO in whole animals was found by culturing the nematode on agar plates seeded with E. coli OP50 and containing the specific MAO A suicide inhibitor clorgyline or the structurally similar specific MAO B inhibitor deprenyl. For assays, starved Bristol N2 hermaphrodites from a plate were transferred to give 5 to 6 individuals per test plate. Plates were sealed with paraffin film and incubated at 20°C and monitored for two weeks with a microscope at 100 and 200x power. Clorgyline inhibited growth of the worm at 2.5 x10 -5Mwhile deprenyl showed inhibition at >1 x10 but <1 x10 -3M.The effect with clorgyline expressed at 25 µM is a concentration 2000 times lower than was required to observe behavioral effects in similar incubation studies with exogenously added neurotransmitters (Horvitz et al., '82, Science 216,1014). The high concentration of amines required in these experiments was attributed to impermeability of the worm's cuticle. This suggests that inhibition of growth results from an even lower concentration of clorgyline entering the animal. In vitro, MAO A is rapidly inhibited by 1 x10 -7Mclorgyline or 1 x10 -5Mdeprenyl.
In addition to the observation that worms stopped growing in the presence of clorgyline and bacteria, cessation of pharyngeal pumping was evident. It is possible that inactivation of MAO increases the concentration of octopamine in a neuron that controls inhibition of pharyngeal pumping (Horvitz et al., '82, ibid.). Growth may have ceased due to starvation. Treatment of octopamine-defective and other mutants with clorgyline and other drugs should shed light on this proposal.
These preliminary experiments strongly suggest the C. elegans contains MAO, possibly of the A type, and that the enzyme plays an essential role in either the development or the normal functioning of this simple organism. We now plan to firmly establish the presence of MAO and its type and determine the mechanism of growth arrest as this most likely underlies the role of MAO and its mode of action in C. elegans.