Ca(2+) sparks are fundamental Ca(2+) signaling events arising from ryanodine receptor (RyR) activation, events that relate to contractile and dilatory events in the pulmonary vasculature. Recent studies demonstrate that long-term hypoxia (LTH) can affect pulmonary arterial reactivity in fetal, newborn, and adult animals. Because RyRs are important to pulmonary vascular reactivity and reactivity changes with ontogeny and LTH we tested the hypothesis that RyR-generated Ca(2+) signals are more active before birth and that LTH suppresses these responses. We examined these hypotheses by performing confocal imaging of myocytes in living arteries and by performing wire myography studies. Pulmonary arteries (PA) were isolated from fetal, newborn, or adult sheep that lived at low altitude or from those that were acclimatized to 3,801 m for > 100 days. Confocal imaging demonstrated preservation of the distance between the sarcoplasmic reticulum, nucleus, and plasma membrane in PA myocytes. Maturation increased global Ca(2+) waves and Ca(2+) spark activity, with sparks becoming larger, wider, and slower. LTH preferentially depressed Ca(2+) spark activity in immature pulmonary arterial myocytes, and these sparks were smaller, wider, and slower. LTH also suppressed caffeine-elicited contraction in fetal PA but augmented contraction in the newborn and adult. The influence of both ontogeny and LTH on RyR-dependent cell excitability shed new light on the therapeutic potential of these channels for the treatment of pulmonary vascular disease in newborns as well as adults.

. Preservation of serotonin-mediated contractility in adult sheep pulmonary arteries following long-term high-altitude hypoxia. High Alt. Med. Biol. 12:253-264.-Long-term hypoxia (LTH) can increase serotonin (5-HT) signaling as well as extracellular calcium entry in adult rodent pulmonary arteries (PA), and 5-HT is associated with pulmonary hypertension. Because LTH, 5-HT, and calcium entry are related, we tested the hypothesis that LTH increases 5-HT-mediated PA contractility and associated calcium influx through L-type Ca 2 + channels, nonselective cation channels (NSCC), and reverse-mode sodium-Ca 2 + exchange. We performed wire myography and confocal calcium imaging on pulmonary arteries from adult ewes that lived near sea level or were maintained at high-altitude (3801 m) for *110 days. LTH did not increase the arterial medial wall thickness, nor did it affect the potency or efficacy for 5-HT-induced PA contraction. Ketanserin (100 nM), a 5-HT 2A antagonist, shifted the 5-HT potency to a far greater extent than 1 lM GR-55562, a 5-HT 1B/D inhibitor. These influences were unaffected by LTH. The rank order for reducing 5-

DNA methylation is fundamental for the stability and activity of genomes. Drosophila melanogaster and vertebrates establish a global DNA methylation pattern of their genome during early embryogenesis. Large-scale analyses of DNA methylation patterns have uncovered revealed that DNA methylation patterns are dynamic rather than static and change in a gene-specific fashion during development and in diseased cells. However, the factors and mechanisms involved in dynamic, postembryonic DNA methylation remain unclear. Methylation of lysine 9 in histone H3 (H3-K9) by members of the Su(var)3–9 family of histone methyltransferases (HMTs) triggers embryonic DNA methylation in Arthropods and Chordates. Here, we demonstrate that Drosophila SETDB1 (dSETDB1) can mediate DNA methylation and silencing of genes and retrotransposons. We found that dSETDB1 tri-methylates H3-K9 and binds methylated CpA motifs. Tri-methylation of H3-K9 by dSETDB1 mediates recruitment of DNA methyltransferase 2 (Dnmt2) and Su(var)205, the Drosophila ortholog of mammalian “Heterochromatin Protein 1”, to target genes for dSETDB1. By enlisting Dnmt2 and Su(var)205, dSETDB1 triggers DNA methylation and silencing of genes and retrotransposons in Drosophila cells. DSETDB1 is involved in postembryonic DNA methylation and silencing of Rt1b{} retrotransposons and the tumor suppressor gene retinoblastoma family protein 1 (Rb) in imaginal discs. Collectively, our findings implicate dSETDB1 in postembryonic DNA methylation, provide a model for silencing of the tumor suppressor Rb, and uncover a role for cell type-specific DNA methylation in Drosophila development.

Endothelial cell activation can hyperpolarize vascular myocytes, resulting in myocyte relaxation and vessel dilation. Direct communication by way of myoendothelial junctions (MEJ) made through the internal elastic lamina (IEL) contributes to vasodilatory responses. The function of the MEJ in pulmonary arteries is not completely understood and less is known regarding their function before birth. The fetal lung has little blood flow and endothelium‐dependent relaxation of pulmonary arteries is limited, which lead us to hypothesize that MEJs are underdeveloped in the fetal lung. This hypothesis was tested using laser scanning confocal microscopy techniques and viewing the IEL of pulmonary arteries from fetal and adult sheep. The IEL was viewed using autofluorescence, and MEJ's were visualized using cell mask red fluorescence. The number and size of pores in the IEL were quantified to index potential MEJ sites and this was compared to the number of MEJ sites that stained positive for cell membrane. Preliminary data suggests the size of each IEL pore, total pore area, and percentage of IEL holes with MEJs was greater in the adult. The restricted IEL pore and MEJ formation in the fetus could diminish MEJ –dependent myocyte hyperpolarization and resultant vasodilation. This could contribute to the high vascular resistance in the fetal pulmonary vasculature. Support from HHMI, NIH and NSF.

Phosphodiesterase (PDE) inhibition can elevate cAMP, dilate pulmonary arteries (PA) and is a therapeutic target for persistent pulmonary hypertension of the newborn. To test whether long term hypoxia (LTH) impairs postnatal maturation of cAMP‐mediated vasodilation and associated Ca2+ signals we reduced cAMP degradation by treating PAs with a non‐selective PDE inhibitor (IBMX). Wire‐myography and confocal Ca2+ imaging were performed on PA isolated from fetal, newborn, or adult sheep that lived at low (720 m) or high altitude (3,801 m) for >;100 days. IBMX caused a dose‐dependent relaxation of serotonin‐constricted PA from all groups and reduced Ca2+ wave amplitude as well as other spatial and temporal components to the Ca2+ signals in newborns but not in fetuses. In arteries from adult normoxic sheep, IBMX impaired spatial and temporal Ca2+ signals. Yet, IBMX also reduced Ca2+ wave amplitude in arteries of LTH adults. LTH increased Ca2+ wave amplitudes in newborns and modified spatial and temporal components to the waves, but failed to alter signals in the fetus. With regards to development, Ca2+ wave amplitudes were elevated in the newborn relative to the fetus and the adult, while spatial and temporal aspects were unchanged. These results suggest that cAMP dilates immature and adult pulmonary arteries, in part, by depressing Ca2+ signals. NSF MRI923559, NIH R03HD69746, R01HD31226, R01HD3807, P20MD6988