The Receptor-Bound Guanylyl Cyclase DAF-11 Is the Mediator of Hydrogen Peroxide-Induced Cgmp Increase in Caenorhabditis elegans

Abstract

Adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) are well-studied second messengers that transmit extracellular signals into mammalian cells, with conserved functions in various other species such as Caenorhabditis elegans (C. elegans). cAMP is generated by adenylyl cyclases, and cGMP is generated by guanylyl cyclases, respectively. Studies using C. elegans have revealed additional roles for cGMP signaling in lifespan extension. For example, mutants lacking the function of a specific receptor-bound guanylyl cyclase, DAF-11, have an increased life expectancy. While the daf-11 phenotype has been attributed to reductions in intracellular cGMP concentrations, the actual content of cyclic nucleotides has not been biochemically determined in this system. Similar assumptions were made in studies using phosphodiesterase loss-of-function mutants or using adenylyl cyclase overexpressing mutants. In the present study, cyclic nucleotide regulation in C. elegans was studied by establishing a special nematode protocol for the simultaneous detection and quantitation of cyclic nucleotides. We also examined the influence of reactive oxygen species (ROS) on cyclic nucleotide metabolism and lifespan in C. elegans using highly specific HPLC-coupled tandem mass-spectrometry and behavioral assays. Here, we show that the relation between cGMP and survival is more complex than previously appreciated.

cCMP and cUMP occur in vivo

“…In none of the organs, we could detect cUMP. This could have been due to the high LLOQ for cUMP [13]. In addition, quenching of MS signals by organ matrix components could contribute to our failure to detect cUMP [12].…”

“…cNMP quantitation was performed via HPLC-MS/MS using a QTrap5500 triple quadrupole mass spectrometer (ABSCIEX, Foster City, CA, USA) [5–7,13]. cNMP analysis by HPLC-MS/TOF was performed as described [5].…”

“…Analyst TF 1.5.1 software was used for data calculation. The LLOQ for standard cAMP was 0.04 pmol/sample, for standard cGMP 0.07 pmol per sample, for standard cCMP 0.07 pmol/sample, and for standard cUMP 0.4 pmol per sample [13]. …”

“…The identification of cCMP and cUMP in vivo is critical for establishing their roles as new second messenger molecules, but technically, this is not trivial. Specifically, matrix effects in organ extracts, resulting in signal suppression, are an inherent problem of HPLC-MS/MS studies with complex biological samples [12,13]. Additionally, among all four cNMPs considered here, cUMP is detected with the lowest sensitivity so that low organ cUMP levels are likely below the LLOQ, i.e.…”

“…Additionally, among all four cNMPs considered here, cUMP is detected with the lowest sensitivity so that low organ cUMP levels are likely below the LLOQ, i.e. 0.4 pmol/ sample [13]. As important experimental tool, we used the Pseudomonas aeruginosa NC toxin ExoY that generates large quantities of cUMP and, to a lesser extent, cCMP in various mammalian cells [14].…”