Regulation of guanylyl cyclase activity in the nervous system of Manduca sexta

Abstract

The intracellular messenger cGMP plays an important role in numerous physiological functions. The enzyme responsible for its synthesis is guanylyl cyclase (GC). I have studied regulation of this enzyme using the Manduca sexta response to eclosion hormone (EH) as a model system. Previous evidence has suggested that EH acts through stimulation of an unusual form of GC which is cytoplasmically localized yet insensitive to activation by NO. This hypothesis has been further supported by work done in the present study. I have shown that EH is unable to stimulate receptor GC activity in central nervous tissue homogenates even with inclusion of the cofactor ATP. I have also shown that EH acts to stimulate GC activity in the soluble fraction of central nervous system (CNS) homogenates when applied to the intact abdominal CNS prior to homogenization. Evidence from the use of inhibitors suggest that this activity may be NO-insensitive. A model for EH action has previously been suggested whereby stimulation of soluble GC activity is preceded by generation of a lipid messenger. I have examined the effect of EH on at least ten different potential lipid messengers or messenger precursors and have found no evidence for generation of a lipid messenger in the EH signal-transduction cascade. In order to examine the possibility of finding a novel, NO-insensitive, soluble GC in Manduca sexta. I, with others, have cloned some of the GCs present in the Manduca abdominal CNS. This has resulted in the finding of a novel GC, MsGCI. This clone contains a catalytic domain which is most similar to receptor GCs, but does not contain ligand-binding, transmembrane, or kinase-like domains. It also does not contain the soluble GC residues thought to be involved in heme-binding, nor is it sensitive to stimulation by NO. When expressed in COS-7 cells, MsGCI shows cytoplasmic localization, activity as an oligomer, and high basal activity. Western blot analysis, however, shows the presence of MsGCI in the particulate fraction of abdominal CNS homogenates. These data suggest a new mechanism for regulation of GC activity based on intracellular translocation.