Differential Effects of Cystathionine-γ-lyase-Dependent Vasodilatory H(2)S in Periadventitial Vasoregulation of Rat and Mouse Aortas

Background

Hydrogen sulfide (H₂S) is a potent vasodilator. However, the complex mechanisms of vasoregulation by H₂S are not fully understood. We tested the hypotheses that (1) H₂S exerts vasodilatory effects by opening KCNQ-type voltage-dependent (K_v) K⁺ channels and (2) that H₂S-producing cystathionine-γ-lyase (CSE) in perivascular adipose tissue plays a major role in this pathway.

Methodology/Principal Findings

Wire myography of rat and mouse aortas was used. NaHS and 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) were used as H₂S donors. KCNQ-type K_v channels were blocked by XE991. 4-Propargylglycine (PPG) and ß-cyano-l-alanine (BCA), or 2-(aminooxy)-acetic acid (AOAA) were used as inhibitors of CSE or cystathionine-ß-synthase (CBS), respectively. NaHS and ADTOH produced strong vasorelaxation in rat and mouse aortas, which were abolished by KCNQ channel inhibition with XE991. Perivascular adipose tissue (PVAT) exerted an anticontractile effect in these arteries. CSE inhibition by PPG and BCA reduced this effect in aortas from rats but not from mice. CBS inhibition with AOAA did not inhibit the anticontractile effects of PVAT. XE991, however, almost completely suppressed the anticontractile effects of PVAT in both species. Exogenous l-cysteine, substrate for the endogenous production of H₂S, induced vasorelaxation only at concentrations >5 mmol/l, an effect unchanged by CSE inhibition.

Conclusions/Signficance

Our results demonstrate potent vasorelaxant effects of H₂S donors in large arteries of both rats and mice, in which XE991-sensitive KCNQ-type channel opening play a pivotal role. CSE-H₂S seems to modulate the effect of adipocyte-derived relaxing factor in rat but not in mouse aorta. The present study provides novel insight into the interaction of CSE-H₂S and perivascular adipose tissue. Furthermore, with additional technical advances, a future clinical approach targeting vascular H₂S/KCNQ pathways to influence states of vascular dysfunction may be possible.     

Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed.     

Construction and analysis of 2 reciprocal Arabidopsis introgression line populations

Two new large reciprocal sets of introgression lines (ILs) were created between the Arabidopsis accessions Col-0 and C24. In both sets (78 ILs with Col-0 background and 62 ILs with C24 background), the donor segments cover almost the entire genome with an average substitution size of 18.3 cM. In addition to the basic sets of ILs, further subILs were developed for 2 genomic regions allowing better mapping resolution. SubILs carrying donor segments with candidate genes for flowering time and reduced fertility were used to demonstrate the usefulness of the reciprocal ILs for quantitative trait loci detection and fine mapping. For subIL development at high resolution around the reduced fertility locus, we used modified CelI-based assays in one-well format for both marker development and genotyping. This serves as a very flexible and cost-effective approach.     

Gq-coupled receptors as mechanosensors mediating myogenic vasoconstriction

Despite the central physiological function of the myogenic response, the underlying signalling pathways and the identity of mechanosensors in vascular smooth muscle (VSM) are still elusive. In contrast to present thinking, we show that membrane stretch does not primarily gate mechanosensitive transient receptor potential (TRP) ion channels, but leads to agonist-independent activation of G(q/11)-coupled receptors, which subsequently signal to TRPC channels in a G protein- and phospholipase C-dependent manner. Mechanically activated receptors adopt an active conformation, allowing for productive G protein coupling and recruitment of beta-arrestin. Agonist-independent receptor activation by mechanical stimuli is blocked by specific antagonists and inverse agonists. Increasing the AT(1) angiotensin II receptor density in mechanically unresponsive rat aortic A7r5 cells resulted in mechanosensitivity. Myogenic tone of cerebral and renal arteries is profoundly diminished by the inverse angiotensin II AT(1) receptor agonist losartan independently of angiotensin II (AII) secretion. This inhibitory effect is enhanced in blood vessels of mice deficient in the regulator of G-protein signalling-2. These findings suggest that G(q/11)-coupled receptors function as sensors of membrane stretch in VSM cells.     

Regulation of oxidative phosphorylation,the mitochondrial membrane potential,and their role in human disease

Thirty years after Peter Mitchell was awarded the Nobel Prize for the chemiosmotic hypothesis, which links the mitochondrial membrane potential generated by the proton pumps of the electron transport chain to ATP production by ATP synthase, the molecular players involved once again attract attention. This is so because medical research increasingly recognizes mitochondrial dysfunction as a major factor in the pathology of numerous human diseases, including diabetes, cancer, neurodegenerative diseases, and ischemia reperfusion injury. We propose a model linking mitochondrial oxidative phosphorylation (OxPhos) to human disease, through a lack of energy, excessive free radical production, or a combination of both. We discuss the regulation of OxPhos by cell signaling pathways as a main regulatory mechanism in higher organisms, which in turn determines the magnitude of the mitochondrial membrane potential: if too low, ATP production cannot meet demand, and if too high, free radicals are produced. This model is presented in light of the recently emerging understanding of mechanisms that regulate mammalian cytochrome c oxidase and its substrate cytochrome c as representative enzymes for the entire OxPhos system.     

Betacellulin overexpression in the mouse ovary leads to MAPK3/MAPK1 hyperactivation and reduces litter size by impairing fertilization

The epidermal growth factor receptor (EGFR) and its ligands are emerging as key molecules in regulating female reproduction. Here, we used a transgenic mouse model to evaluate whether and at which level of the reproduction cascade higher-than-normal levels of the EGFR ligand betacellulin (BTC) in the reproductive organs affect fertility. Western blots and immunohistochemistry revealed increased BTC levels in uterus and ovaries from transgenic females, particularly evident in granulosa cells of antral follicles. Onset of puberty, estrous cyclicity, and the anatomy and histology of reproductive organs at puberty were not altered as compared to control females. Fertility tests revealed a reduction (~50%) in litter size as the major reproductive deficit of transgenic females. Embryo implantation was delayed in transgenic females, but this was not the reason for the reduced litter size. Transgenic females produced a normal number of oocytes after natural ovulation. The in vivo fertilization rate was significantly reduced in untreated transgenic females but returned to normal levels after superovulation. Impaired oocyte fertilization in the absence of superovulation treatment was associated with MAPK3/MAPK1 hyperactivation in BTC transgenic ovaries, whereas similar levels of MAPK3/MAPK1 activation were detected in transgenic and control ovaries after superovulation treatment. Thus, tight regulation of MAPK3/MAPK1 activity appears to be essential for appropriate granulosa cell function during oocyte maturation. Our study identified hitherto unknown effects of BTC overabundance in reproduction and suggests BTC as a novel candidate protein for the modulation of fertility.     

Partitioning direct and indirect human-induced effects on carbon sequestration of managed coniferous forests using model simulations and forest inventories

Temperate forest ecosystems have recently been identified as an important net sink in the global carbon budget. The factors responsible for the strength of the sinks and their permanence, however, are less evident. In this paper, we quantify the present carbon sequestration in Thuringian managed coniferous forests. We quantify the effects of indirect human‐induced environmental changes (increasing temperature, increasing atmospheric CO₂ concentration and nitrogen fertilization), during the last century using BIOME‐BGC, as well as the legacy effect of the current age‐class distribution (forest inventories and BIOME‐BGC). We focused on coniferous forests because these forests represent a large area of central European forests and detailed forest inventories were available. The model indicates that environmental changes induced an increase in biomass C accumulation for all age classes during the last 20 years (1982–2001). Young and old stands had the highest changes in the biomass C accumulation during this period. During the last century mature stands (older than 80 years) turned from being almost carbon neutral to carbon sinks. In high elevations nitrogen deposition explained most of the increase of net ecosystem production (NEP) of forests. CO₂ fertilization was the main factor increasing NEP of forests in the middle and low elevations. According to the model, at present, total biomass C accumulation in coniferous forests of Thuringia was estimated at 1.51 t C ha^?1 yr^?1 with an averaged annual NEP of 1.42 t C ha^?1 yr^?1 and total net biome production of 1.03 t C ha^?1 yr^?1 (accounting for harvest). The annual averaged biomass carbon balance (BCB: biomass accumulation rate‐harvest) was 1.12 t C ha^?1 yr^?1 (not including soil respiration), and was close to BCB from forest inventories (1.15 t C ha^?1 yr^?1). Indirect human impact resulted in 33% increase in modeled biomass carbon accumulation in coniferous forests in Thuringia during the last century. From the forest inventory data we estimated the legacy effect of the age‐class distribution to account for 17% of the inventory‐based sink. Isolating the environmental change effects showed that these effects can be large in a long‐term, managed conifer forest.