No abstract

The Drosophila melanogaster genome contains 5 genes that code for soluble guanylyl cyclase subunits. Two of these genes code for subunits, Gycα-99B and Gycβ-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 Km, 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.Abbreviation:ORFopen reading frameUTRuntranslated region

No abstract

SUMMARY 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.

No abstract

Nicotinic acetylcholine receptors (nAChRs) in insects are neuron-specific oligomeric proteins essential for the central transmission of sensory information. Little is known about their subunit composition because it is difficult to express functional insect nAChRs in heterologous systems. As an alternative approach we have examined the native expression of two subunits in neurons of the nicotinic-resistant, tobacco-feeding insect Manduca sexta. Both the alpha-subunit MARA1 and the beta-subunit MARB can be detected by in situ hybridization in the majority of cultured neurons with an overlapping, but not identical, distribution. Changes in intracellular Ca(2+) evoked by nicotinic stimulation are more strongly correlated to the expression of MARA1 than MARB and are independent of cell size. Unlike the previously reported critical role of MARA1 in mediating nicotinic Ca(2+) responses, down-regulation of MARB by RNA interference (RNAi) did not reduce the number of responding neurons or the size of evoked responses, suggesting that additional subunits remain to be identified in Manduca.

Background and Objective:The objective of this study was to investigate the effects of resveratrol (RES) on Arachidonic acid (AA) metabolism in the kidney and its effect on arterial blood pressure, using spontaneously hypertensive rats (SHR) as a model system. Methods: Rats were exposed to either drinking water alone (control) or RES (20 or 40 mg kgG 1 ) added to drinking water for 7 weeks. Mean Arterial Pressure (MAP) was measured at 7-day intervals throughout the study. At the end of treatment, rats were euthanized, followed by preparation of kidney microsomes to measure enzymes involved in regulation of vasoactive metabolites: CYP4A, the key enzyme in the formation of 20-hydroxyeicosatetraenoic acid and the soluble epoxide hydrolase, which is responsible for the degradation of the vasodilator metabolites such as epoxyeicosatetraenoic acids. Effect of RES on kidney expression of CYP4A was also investigated by immunoblotting. Results: Treatment with RES resisted the progressive rise in MAP in the developing SHR in a dose-dependent manner. Consistent with these data, RES treatment led to significant reductions in both, the expression and activity of renal CYP4A isozymes, as well as the activity of soluble epoxide hydrolase (sEH). Conclusion: The presented studies show that RES modulates the metabolism of AA by both P450 enzymes and sEH in SHR rats, which may represent a novel mechanism by which RES protects SHR rats against the progressive rise in blood pressure.

Soluble guanylyl cyclase (sGC) is probably the most prevalent target for NO in almost all species and tissues (Lucas et al., 2000). The native enzyme is usually a heterodimer (see below for exceptions to this) containing a single heme group (Lucas et al., 2000). In mammals, four subunits have been identified: α1, α2, β1 and β2. The major functional enzyme is the α1/β1 heterodimer (Lucas et al., 2000). The α2 is very similar in sequence to the α1 subunit and the α2/β1 heterodimer has similar properties to the α1/β1 enzyme (Russworm et al., 1998, Gibb et al., 2003). One significant difference is that the α2 subunit interacts with the scaffold protein PSD95 and hence likely has a different subcellular distribution (Russworm et al., 2001). All four mammalian subunits are homologous proteins with a similar arrangement of functional domains (Figure 1). The subunits can be divided into a C-terminal catalytic domain that catalyses the conversion of GTP to cGMP and an N-terminal regulatory domain that functions as a heme binding region and is required for NO activation of the enzyme (Figure 1). A variety of studies have identified several residues in each of these domains that are required for function and are indicated in Figure 1. The α1/β1 heterodimer binds a single heme group per heterodimer and the β1 subunit is primarily responsible for this interaction (Lucas et al., 2000). The Fe 2+ ion in the center of the heme group interacts with His105 (identified as H105 in Figure 1) of the β1 subunit (Zhao et al., 1998). The heme group interacts with three residues in the β1 subunit via hydrogen bonds that form the YXS/TXR motif (Schmidt et al., 2004). In addition, two cysteines (at positions C78 and C214 in the rat β1) subunit that are necessary for NO activation (Freibe et al., 1997). The relative positions of these residues are shown in Figure 1. Modeling of the catalytic domain of sGC, based on the crystal structure of the catalytic domain of mammalian adenylyl cyclase, predicts that there are 17 residues that contact the GTP substrate (Liu et al., 1997). The active site is at the interface between the two subunits, so each subunit provides a subset of specific residues (10 for the β subunit and 7 for the α subunit, which are marked on the catalytic domain in Figure 1). This model predicts that a single GTP molecule will bind per heterodimer (Liu et al., 1997). The β2 subunit has quite different functional characteristics and appears to be a member of a different subfamily of sGC subunits, which we have termed the atypical sGCs (Morton, 2004a). Although the β2 subunit will form active heterodimers with both the α1 and α2, it has the unusual property that it is also active as a homodimer (Koglin et al., 2001; Gibb et al., 2003). Each of these enzymes are sensitive to NO, but they are all less potently activated by NO compared to the α1/β1 and α2/β1 heterodimers (Gibb et al., 2003). The regulatory domain of the β2 subunit contains His104, Cys78, Cys214 and the YXS/TXR motif (Figure 1), which suggests that the α1/β2 and α2/...