Melanie Philipp

Activation of α2B-Adrenoceptors mediates the cardiovascular effects of etomidate

Background The intravenous anesthetic etomidate exhibits structural similarities to specific &agr;2-adrenoceptor agonists of the type such as dexmedetomidine. The current study was performed to elucidate the possible interaction of etomidate with &agr;2-adrenoceptors in mice lacking individual &agr;2-adrenoceptor subtypes (&agr;2-KO). Methods Sedative and cardiovascular responses to etomidate and the &agr;2-agonist, dexmedetomidine, were determined in mice deficient in &agr;2-receptor subtypes. Inhibition of binding of the &agr;2-receptor antagonist [3H]RX821002 to recombinant &agr;2-receptors by etomidate was tested in human embryonic kidney (HEK293) cells in vitro. Results In vivo, loss and recovery of the righting reflex required similar times after intraperitoneal injection of etomidate in wild-type and in &agr;2A-receptor–deficient mice, indicating that the hypnotic effect of etomidate in mice does not require the &agr;2A-receptor subtype. Intravenous injection of etomidate resulted in a transient increase (duration 2.4 ± 0.2 min) in arterial blood pressure in wild-type mice (17 ± 3 mmHg). Etomidate did not affect blood pressure in &agr;2B-KO or &agr;2AB-KO mice. In membranes from HEK293 cells transfected with &agr;2-receptors, etomidate inhibited binding of the &agr;2-antagonist, [3H]RX821002, with higher potency from &agr;2B- and &agr;2C-receptors than from &agr;2A-receptors (Ki &agr;2A 208 &mgr;m , &agr;2B 26 &mgr;m, &agr;2C 56 &mgr;m). In &agr;2B-receptor–expressing HEK293 cells, etomidate rapidly increased phosphorylation of the extracellular signal-related kinases ERK1/2. Conclusions These results indicate that etomidate acts as an agonist at &agr;2-adrenoceptors, which appears in vivo primarily as an &agr;2B-receptor–mediated increase in blood pressure. This effect of etomidate may contribute to the cardiovascular stability of patients after induction of anesthesia with etomidate.

All three α2-adrenoceptor types serve as autoreceptors in postganglionic sympathetic neurons

Postganglionic sympathetic neurons and brain noradrenergic neurons use α2A- and α2C-adrenoceptors as presynaptic autoreceptors. The present experiments were carried out in order to see whether they possess presynaptic α2B-autoreceptors as well. Pieces of atria, vasa deferentia, the occipito-parietal cortex and the hippocampus were prepared from either wildtype (WT) mice or mice in which both the α2A- and the α2C-adrenoceptor gene had been disrupted (α2ACKO). The pieces were incubated with 3H-noradrenaline and then superfused and stimulated electrically. In a first series of experiments, single pulses or brief, autoinhibition-poor pulse trains were used for stimulation. The α2-adrenoceptor agonist UK 14,304 (brimonidine) reduced the evoked overflow of tritium in all four tissues from WT mice but did not change it in any tissue from α2ACKO mice. A different pattern was obtained with medetomidine as α2 agonist. Like UK 14,304, medetomidine reduced the evoked overflow of tritium in all four tissues from WT mice and did not affect overflow in brain slices from α2ACKO mice; however, in contrast to UK 14,304, medetomidine reduced evoked overflow also in atrial and vas deferens pieces from α2ACKO mice, although with a lower maximum and potency than in WT preparations. The α-adrenoceptor antagonists rauwolscine, phentolamine, prazosin, spiroxatrine and WB 4101 shifted the concentration-response curve of medetomidine in α2ACKO atria and vasa deferentia to the right. The pKd values of the five antagonists against medetomidine in α2ACKO atria and vasa deferentia correlated with pKd values at prototypical α2B radioligand binding sites but not at α2A or α2C binding sites. In a second series of experiments, autoinhibition-rich pulse trains were used for stimulation. Under these conditions, rauwolscine and phentolamine increased the evoked overflow of tritium from α2ACKO atrial and vas deferens pieces but not from α2ACKO brain slices. The increase was smaller (by 40% in atria and by 70% in the vas deferens) than previously observed in WT preparations (by 200–400%). In a last series of experiments, mRNA for the α2B-adrenoceptor was demonstrated by RT-PCR in thoracolumbar sympathetic ganglia from WT, α2AKO, α2CKO and α2ACKO mice but not from α2BKO mice. The results show that brain noradrenergic neurons express only α2A- and α2C-adrenoceptors as autoreceptors. Postganglionic sympathetic neurons, however, can express α2B-adrenoceptors as presynaptic autoreceptors as well. The α2B-autoreceptors are activated by medetomidine but not by UK 14,304. They are also activated by previously released noradrenaline. The two-α2-autoreceptor hypothesis has to be replaced by a three-autoreceptor hypothesis for postganglionic sympathetic neurons.

Mutations in SPRTN cause early onset hepatocellular carcinoma, genomic instability and progeroid features

Age-related degenerative and malignant diseases represent major challenges for health care systems. Elucidation of the molecular mechanisms underlying carcinogenesis and age-associated pathologies is thus of growing biomedical relevance. We identified biallelic germline mutations in SPRTN (also called C1orf124 or DVC1) in three patients from two unrelated families. All three patients are affected by a new segmental progeroid syndrome characterized by genomic instability and susceptibility toward early onset hepatocellular carcinoma. SPRTN was recently proposed to have a function in translesional DNA synthesis and the prevention of mutagenesis. Our in vivo and in vitro characterization of identified mutations has uncovered an essential role for SPRTN in the prevention of DNA replication stress during general DNA replication and in replication-related G2/M-checkpoint regulation. In addition to demonstrating the pathogenicity of identified SPRTN mutations, our findings provide a molecular explanation of how SPRTN dysfunction causes accelerated aging and susceptibility toward carcinoma.

Placental alpha(2)-adrenoceptors control vascular development at the interface between mother and embryo.

A substantial percentage of human pregnancies are lost as spontaneous abortions after implantation. This is often caused by an inadequately developed placenta. Proper development of the placental vascular system is essential to nutrient and gas exchange between mother and developing embryo. Here we show that α2-adrenoceptors, which are activated by adrenaline and noradrenaline, are important regulators of placental structure and function. Mice with deletions in the genes encoding α2A-, α2B- and α2C-adrenoceptors died between embryonic days 9.5 and 11.5 from a severe defect in yolk-sac and placenta development. In wildtype placentae, α2-adrenoceptors are abundantly expressed in giant cells, which secrete angiogenic factors to initiate development of the placental vascular labyrinth. In placentae deficient in α2A-, α2B- and α2C-adrenoceptors, the density of fetal blood vessels in the labyrinth was markedly lower than normal, leading to death of the embryos as a result of reduced oxygen and nutrient supply. Basal phosphorylation of the extracellular signal–regulated kinases ERK1 and ERK2 was also lower than normal, suggesting that activation of the mitogen-activated protein kinase (MAP kinase) pathway by α2-adrenoceptors is required for placenta and yolk-sac vascular development. Thus, α2-adrenoceptors are essential at the placental interface between mother and embryo to establish the circulatory system of the placenta and thus maintain pregnancy.

Genetic Dissection of α2-Adrenoceptor Functions in Adrenergic versus Nonadrenergic Cells

α2-Adrenoceptors mediate diverse functions of the sympathetic system and are targets for the treatment of cardiovascular disease, depression, pain, glaucoma, and sympathetic activation during opioid withdrawal. To determine whether α2-adrenoceptors on adrenergic neurons or α2-adrenoceptors on nonadrenergic neurons mediate the physiological and pharmacological responses of α2-agonists, we used the dopamine β-hydroxylase (Dbh) promoter to drive expression of α2A-adrenoceptors exclusively in noradrenergic and adrenergic cells of transgenic mice. Dbh-α2A transgenic mice were crossed with double knockout mice lacking both α2A- and α2C-receptors to generate lines with selective expression of α2A-autoreceptors in adrenergic cells. These mice were subjected to a comprehensive phenotype analysis and compared with wild-type mice, which express α2A- and α2C-receptors in both adrenergic and nonadrenergic cells, and α2A/α2C double-knockout mice, which do not express these receptors in any cell type. We were surprised to find that only a few functions previously ascribed to α2-adrenoceptors were mediated by receptors on adrenergic neurons, including feedback inhibition of norepinephrine release from sympathetic nerves and spontaneous locomotor activity. Other agonist effects, including analgesia, hypothermia, sedation, and anesthetic-sparing, were mediated by α2-receptors in nonadrenergic cells. In dopamine β-hydroxylase knockout mice lacking norepinephrine, the α2-agonist medetomidine still induced a loss of the righting reflex, confirming that the sedative effect of α2-adrenoceptor stimulation is not mediated via autoreceptor-mediated inhibition of norepinephrine release. The present study paves the way for a revision of the current view of the α2-adrenergic receptors, and it provides important new considerations for future drug development.

Smoothened Signaling in Vertebrates Is Facilitated by a G Protein-coupled Receptor Kinase

Smoothened, a heptahelical membrane protein, functions as the transducer of Hedgehog signaling. The kinases that modulate Smoothened have been thoroughly analyzed in flies. However, little is known about how phosphorylation affects Smoothened in vertebrates, mainly, because the residues, where Smoothened is phosphorylated are not conserved from Drosophila to vertebrates. Given its molecular architecture, Smoothened signaling is likely to be regulated in a manner analogous to G protein-coupled receptors (GPCRs). Previously, it has been shown, that arrestins and GPCR kinases, (GRKs) not only desensitize G protein-dependent receptor signaling but also function as triggers for GPCR trafficking and formation of signaling complexes. Here we describe that a GRK contributes to Smoothened-mediated signaling in vertebrates. Knockdown of the zebrafish homolog of mammalian GRK2/3 results in lowered Hedgehog transcriptional responses, impaired muscle development, and neural patterning. Results obtained in zebrafish are corroborated both in cell culture, where zGRK2/3 phosphorylates Smoothened and promotes Smoothened signal transduction and in mice where deletion of GRK2 interferes with neural tube patterning. Together, these data suggest that a GRK functions as a vertebrate kinase for Smoothened, promoting Hedgehog signal transduction during early development.

G protein-coupled receptor kinases phosphorylate LRP6 in the Wnt pathway

Wnt ligands conduct their functions in canonical Wnt signaling by binding to two receptors, the single transmembrane low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) and seven transmembrane (7TM) Frizzled receptors. Subsequently, phosphorylation of serine/threonine residues within five repeating signature PPPSP motifs on LRP6 is responsible for LRP6 activation. GSK3β, a cytosolic kinase for phosphorylation of a downstream effector β-catenin, was proposed to participate in such LRP6 phosphorylation. Here, we report a new class of membrane-associated kinases for LRP6 phosphorylation. We found that G protein-coupled receptor kinases 5 and 6 (GRK5/6), traditionally known to phosphorylate and desensitize 7TM G protein-coupled receptors, directly phosphorylate the PPPSP motifs on single transmembrane LRP6 and regulate Wnt/LRP6 signaling. GRK5/6-induced LRP6 activation is inhibited by the LRP6 antagonist Dickkopf. Depletion of GRK5 markedly reduces Wnt3A-stimulated LRP6 phosphorylation in cells. In zebrafish, functional knock-down of GRK5 results in reduced Wnt signaling, analogous to LRP6 knock-down, as assessed by decreased abundance of β-catenin and lowered expression of the Wnt target genes cdx4, vent, and axin2. Expression of GRK5 rescues the diminished β-catenin and axin2 response caused by GRK5 depletion. Thus, our findings identify GRK5/6 as novel kinases for the single transmembrane receptor LRP6 during Wnt signaling.

Quantitative Label-Free Phosphoproteomics Strategy for Multifaceted Experimental Designs

Protein phosphorylation is a critical regulator of signaling in nearly all eukaryotic cellular pathways and dysregulated phosphorylation has been implicated in an array of diseases. The majority of MS-based quantitative phosphorylation studies are currently performed from transformed cell lines because of the ability to generate large amounts of starting material with incorporated isotopically labeled amino acids during cell culture. Here we describe a general label-free quantitative phosphoproteomic strategy capable of directly analyzing relatively small amounts of virtually any biological matrix, including human tissue and biological fluids. The strategy utilizes a TiO2 enrichment protocol in which the selectivity and recovery of phosphopeptides were optimized by assessing a twenty-point condition matrix of binding modifier concentrations and peptide-to-resin capacity ratios. The quantitative reproducibility of the TiO2 enrichment was determined to be 16% RSD through replicate enrichments of a wild-type Danio rerio (zebrafish) lysate. Measured phosphopeptide fold-changes from alpha-casein spiked into wild-type zebrafish lysate backgrounds were within 5% of the theoretical value. Application to a morpholino induced knock-down of G protein-coupled receptor kinase 5 (GRK5) in zebrafish embryos resulted in the quantitation of 719 phosphorylated peptides corresponding to 449 phosphorylated proteins from 200 μg of zebrafish embryo lysates.

RabGDI controls axonal midline crossing by regulating Robo1 surface expression

BackgroundAxons navigate to their future synaptic targets with the help of choice points, intermediate targets that express axon guidance cues. Once they reach a choice point, axons need to switch their response from attraction to repulsion in order to move on with the next stage of their journey. The mechanisms underlying the change in axonal responsiveness are poorly understood. Commissural axons become sensitive to the repulsive activity of Slits when they cross the ventral midline of the CNS. Responsiveness to Slits depends on surface expression of Robo receptors. In Drosophila, Commissureless (Comm) plays a crucial regulatory role in midline crossing by keeping Robo levels low on precommissural axons. Interestingly, to date no vertebrate homolog of comm has been identified. Robo3/Rig1 has been shown to control Slit sensitivity before the midline, but without affecting Robo1 surface expression.ResultsWe had identified RabGDI, a gene linked to human mental retardation and an essential component of the vesicle fusion machinery, in a screen for differentially expressed floor-plate genes. Downregulation of RabGDI by in ovo RNAi caused commissural axons to stall in the floor plate, phenocopying the effect observed after downregulation of Robo1. Conversely, premature expression of RabGDI prevented commissural axons from entering the floor plate. Furthermore, RabGDI triggered Robo1 surface expression in cultured commissural neurons. Taken together, our results identify RabGDI as a component of the switching mechanism that is required for commissural axons to change their response from attraction to repulsion at the intermediate target.ConclusionRabGDI takes over the functional role of fly Comm by regulating the surface expression of Robo1 on commissural axons in vertebrates. This in turn allows commissural axons to switch from attraction to repulsion at the midline of the spinal cord.

Hedgehog Signaling: Is Smo a G Protein-Coupled Receptor?

The Hedgehog signal transducer Smoothened is structurally similar to G protein-coupled receptors. Now there is direct evidence that Smoothened relies on heterotrimeric G proteins in order to transduce the Hedgehog signal.