RESUMO
Responses to hypoxia and hyperoxia depend critically on the ability of the animal to detect changes in O2 levels. However, it has only been recently that an O2-sensing system has been identified in invertebrates. Evidence is accumulating that this molecular O2 sensor is, surprisingly, a class of soluble guanylyl cyclase (sGC) known as atypical sGCs. It has long been known that the conventional sGC alpha and beta subunits form heterodimeric enzymes that are potently activated by NO, but do not bind O2. By contrast, the Drosophila melanogaster atypical sGC subunits, Gyc-88E, Gyc-89Da and Gyc-89Db, are only slightly sensitive to NO, but are potently activated under hypoxic conditions. Here we review evidence that suggests that the atypical sGCs can function as molecular O2 sensors mediating behavioral responses to hypoxia. Sequence comparisons of other predicted O2-sensitive sGCs suggest that most, if not all, insects express two heterodimeric sGCs; an NO-sensitive isoform and a separate O2-sensitive isoform. Expression data and recent experiments that block the function of cells that express the atypical sGCs and experiments that reduce the cGMP levels in these cells also suggest a role in behavioral responses to sweet tastants.
Assuntos
Comportamento Alimentar/fisiologia , Guanilato Ciclase/fisiologia , Insetos/enzimologia , Oxigênio/fisiologia , Sequência de Aminoácidos , Animais , Drosophila/enzimologia , Drosophila/genética , Guanilato Ciclase/química , Insetos/genética , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
The Drosophila melanogaster genome contains 5 genes that code for soluble guanylyl cyclase subunits. Two of these genes code for subunits, Gycalpha-99B and Gycbeta-100B, which form a conventional NO-sensitive guanylyl cyclase and the other three code for atypical subunits, Gyc-88E, Gyc-89Da and Gyc-89Db. The properties and distribution of Gyc-88E and Gyc-89Db have previously been described and here Gyc-89Da is described. Gyc-89Da only forms an active guanylyl cyclase when co-expressed with Gyc-88E. The three atypical subunits probably form two different heterodimers in vivo: Gyc-88E/89Da and Gyc-88E/89Db. Both of these heterodimers were slightly stimulated by NO donors and Gyc-88E/89Da showed a greater activation by Mn2+, with an increase in Vmax and a decrease in K(m), compared to Gyc-88E/89Db. Both Gyc-88E/89Da and Gyc-88E/89Db were expressed in neurons in both the peripheral and central nervous system. Although all three heterodimeric soluble guanylyl cyclases in D. melanogaster can be activated by NO and inhibited by ODQ, the atypical enzymes can be distinguished from the conventional soluble guanylyl cyclase by their sensitivity to the NO-independent activators YC-1 and BAY 41-2272, which will only activate the conventional enzyme.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/enzimologia , Regulação Enzimológica da Expressão Gênica , Guanilato Ciclase/biossíntese , Guanilato Ciclase/genética , Receptores de Superfície Celular/fisiologia , Animais , Células COS , Chlorocebus aethiops , Clonagem Molecular/métodos , Primers do DNA/química , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/efeitos dos fármacos , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Cistos Glanglionares/ultraestrutura , Perfilação da Expressão Gênica , Ordem dos Genes , Guanilato Ciclase/efeitos dos fármacos , Guanilato Ciclase/fisiologia , Hibridização In Situ/métodos , Estágios do Ciclo de Vida/fisiologia , Doadores de Óxido Nítrico/farmacologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de TempoRESUMO
Insect ecdysis is a precisely coordinated series of behavioral and hormonal events that occur at the end of each molt. A great deal is known about the hormonal events that underlie this process, although less is known about the neuronal circuitry involved. In this study we identified two populations of neurons that are required for larval and adult ecdyses in the fruit fly, Drosophila melanogaster (Meigen). These neurons were identified by using the upstream region of two genes that code for atypical soluble guanylyl cyclases to drive tetanus toxin in the neurons that express these cyclases to block their synaptic activity. Expression of tetanus toxin in neurons that express Gyc-89Da blocked adult eclosion whereas expression of tetanus toxin in neurons that express Gyc-89Db prevented the initiation of the first larval ecdysis. Expression of tetanus toxin in the Gyc-89Da neurons also resulted in about 50% lethality just prior to pupariation; however, this was probably due to suffocation in the food as lethality was prevented by stopping the larvae from burrowing deep within the food. This result is consistent with our model that the atypical soluble guanylyl cyclases can act as molecular oxygen detectors. The expression pattern of these cyclases did not overlap with any of the neurons containing peptides known to regulate ecdysis and eclosion behaviors. By using the conditional expression of tetanus toxin we were also able to demonstrate that synaptic activity in the Gyc-89Da and Gyc-89Db neurons is required during early adult development for adult eclosion.