Infertility and Male Mating Behavior Deficits Associated With Pde1c in Drosophila melanogaster

Pde1c is a calcium/calmodulin-regulated, dual-specificity cyclic nucleotide phosphodiesterase. We have used a transposon insertion line to investigate the physiological function of Pde1c in Drosophila melanogaster and to show that the insertion leads to male sterility and male mating behavior defects that include reduced copulation rates. Sterility appears to be primarily due to elimination of sperm from the female reproductive system. The male mating behavior defects were fully rescued by expression of exogenous Pde1c under the control of either a Pde1c or a pan-neuronal promoter, whereas the sterility could be only partially rescued by expression of exogenous Pde1c under the control of these promoters. We also show that Pde1c has a male-specific expression pattern in the CNS with an increased number of Pde1c-expressing neurons in the abdominal ganglion in males.THE cyclic nucleotides, cyclic AMP (cAMP) and cyclic GMP (cGMP), have been known for many years to regulate a wide variety of physiological processes in all animals (e.g., Siegel et al. 1994). Similarly, there is a large body of research focused on an understanding of the structure and regulation of the enzymes that synthesize cAMP and cGMP, the adenylyl and guanylyl cyclases, respectively. Although the concentrations of cyclic nucleotides within a cell are regulated by both their synthesis and their degradation, less attention has been devoted to the function and regulation of the enzymes that break down cyclic nucleotides, the phosphodiesterases (PDEs).Mammals have >20 genes that code for cyclic nucleotide PDEs, which have been subdivided into 11 families on the basis of their sequences, substrate specificities, and regulatory properties (Conti and Beavo 2007). Insects also have a wide variety of PDEs with Drosophila melanogaster containing 6 genes that code for cyclic nucleotide PDEs (Morton and Hudson 2002; Day et al. 2005). On the basis of their sequence similarity to the mammalian PDEs, these 6 genes have been classified into 6 of the 11 families: Pde1c, Pde4, Pde6, Pde8, Pde9, and Pde11 (Day et al. 2005). When their biochemical properties have been investigated, they match well with other members of the same family (Day et al. 2005).Despite the importance of cyclic nucleotides in insect physiology, only one of the Drosophila PDEs has been associated with a mutant phenotype. This gene, Pde4, also known as dunce, was one of the first learning and memory mutants discovered (Byers et al. 1981; Davis et al. 1995). In this study, we have investigated the phenotypes associated with reduced expression of Pde1c, a PDE that has dual specificity for both cAMP and cGMP and that is stimulated in the presence of calcium and calmodulin (Day et al. 2005). Here we show that Pde1c is required for male fertility and male mating behavior. Male sterility appears to be primarily due to females rejecting sperm or failing to store the sperm from mutant males.     

Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation

In vitro studies have used protein markers to distinguish between myogenic cells isolated from fast and slow skeletal muscles. The protein markers provide some support for the hypothesis that satellite cells from fast and slow muscles are different, but the data are equivocal. To test this hypothesis directly, three-dimensional skeletal muscle constructs were engineered from myogenic cells isolated from fast tibialis anterior (TA) and slow soleus (SOL) muscles of rats and functionality was tested. Time to peak twitch tension (TPT) and half relaxation time (RT_1/2) were 30% slower in constructs from the SOL. The slower contraction and relaxation times for the SOL constructs resulted in left shift of the force-frequency curve compared with those from the TA. Western blot analysis showed a 60% greater quantity of fast myosin heavy chain in the TA constructs. 14 days of chronic low-frequency electrical stimulation resulted in a 15% slower TPT and a 14% slower RT_1/2, but no change in absolute force production in the TA constructs. In SOL constructs, slow electrical stimulation resulted in an 80% increase in absolute force production with no change in TPT or RT_1/2. The addition of cyclosporine A did not prevent the increase in force in SOL constructs after chronic low-frequency electrical stimulation, suggesting that calcineurin is not responsible for the increase in force. We conclude that myogenic cells associated with a slow muscle are imprinted to produce muscle that contracts and relaxes slowly and that calcineurin activity cannot explain the response to a slow pattern of electrical stimulation. tissue engineering; calcineurin; electrical stimulation; engineered muscle; bioreactors     

Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis

Prior studies have shown that fluid secretions from airway submucosal glands in cystic fibrosis (CF) are reduced and hyperviscous, possibly contributing to the pathogenesis of CF airway disease. Because the CF transmembrane conductance regulator (CFTR) protein can transport both chloride and bicarbonate, we investigated whether gland fluid pH is abnormal in early CF, using nasal biopsies from pediatric subjects having minimal CF lung disease. Gland fluid pH, measured in freshly secreted droplets under oil stained with BCECF-dextran, was 6.57 ± 0.09 (mean ± SE) in biopsies from six CF subjects, significantly lower than 7.18 ± 0.06 in eight non-CF biopsies (P < 0.01). To rule out the possibility that the apparent gland fluid hyperacidity in CF results from modification of fluid pH by the airway surface, a microcannulation method was used to measure pH in fluid exiting gland orifices. In pig trachea and human bronchi, gland fluid pH was reduced by up to 0.45 units by CFTR inhibitors, but was not affected by amiloride. Acid base transport in the surface epithelium of pig trachea was studied from pH changes in 300-nl fluid droplets deposited onto the oil-covered airway surface. The droplets had specified ionic composition/pH and/or contained transporter activators/inhibitors. We found evidence for CFTR-dependent bicarbonate transport by the tracheal surface epithelium as well as ATP/histamine-stimulated proton secretion, but not for sodium/proton or chloride/bicarbonate exchange. These results provide evidence for intrinsic hyperacidity in CF gland fluid secretions, which may contribute to CF airway pathology. cystic fibrosis transmembrane conductance regulator; airway; fluorescence microscopy; pH regulation     

Intracellular heterogeneity in adhesiveness of endothelium affects early steps in leukocyte adhesion

Endothelial cell junctions are thought to be preferential sites for transmigration. However, the factors that determine the site of transmigration are not well defined. Our data show that the preferential role of endothelial cell junctions is not limited to transmigration but extends to earlier steps of leukocyte recruitment, such as rolling and arrest. We used primary mouse neutrophils and mouse aortic endothelium in a flow chamber system to compare adhesive interactions near endothelial cell junctions to interactions over endothelial cell centers. We found differences in both rolling velocity and arrest frequency for neutrophils at endothelial cell junctions vs. more central areas of endothelial cells. Differences were governed by adhesion molecule interactions, not local topography. Interestingly, the role of particular adhesion molecules depended on their location on the endothelial cell surface. Although ICAM-1 stabilized and slowed rolling over central areas of the cell, it did not influence rolling velocity over endothelial cell junctions. P-selectin and VCAM-1 were more important for rolling near endothelial cell junctions than E-selectin. This demonstrates that adhesive properties of endothelial cell junctions influence early events in the adhesion cascade, which may help explain how leukocytes are localized to sites of eventual transmigration. endothelial cells; rolling; selectins; integrins