GTP analogs suppress uptake but not transport of d-glucose analogs in Glut1 glucose transporter-expressing Xenopus oocytes

AbstractA Xenopus oocyte expression-co-injection system was used to study the influence of guanine nucleotides on D-glucose uptake. GTP analogs like GTPγS and GppNHp had no effect on 3-O-methylglucose transport determined by zero-trans uptake or equilibrium exchange, but suppressed 2-deoxyglucose uptake into Glutl glucose transporter-expressing oocytes by up to 86%. Both GTP analogs showed concentration dependence of their effectiveness, with GTPγS being more potent than GppNHp. No statistically significant differences were observed between groups of oocytes co-injected with water or GDPβS (250 and 500 μM intracellular concentration). Glut1 transporter expression in plasma membrane was not different between water or GTPγS-co-injected oocytes. Thus, inhibition of hexokinase catalytic activity is the most likely causative factor for down-regulation of 2-deoxyglucose uptake

Inhibition of NF-kappaB by a TAT-NEMO-binding domain peptide accelerates constitutive apoptosis and abrogates LPS-delayed neutrophil apoptosis.

Delivery of biologically active peptides into human polymorphonuclear neutrophils (PMNs) has implications for studying cellular functions and may be therapeutically relevant. The transcription factor nuclear factor-kappaB (NF-kappaB) regulates the expression of multiple genes controlling inflammation, proliferation, and cell survival. PMNs play a crucial role in first-line defense. Targeting NF-kappaB in these cells may promote apoptosis and therefore facilitate resolution of inflammation. We used an 11-amino acid sequence NEMO-binding domain (NBD) that selectively inhibits the IKKgamma (NEMO)/IKKbeta interaction, preventing NF-kappaB activation. An HIV-TAT sequence served as a highly effective transducing shuttle. We show that lipopolysaccharide (LPS), granulocyte-macrophage colony-stimulating factor (GM-CSF), and dexamethasone (DEX) significantly reduced apoptosis after 20 hours. LPS, but not GM-CSF or DEX, activated NF-kappaB as shown by IkappaBalpha degradation, NF-kappaB DNA binding, and transcriptional activity. The TAT-NBD blocked LPS-induced NF-kappaB activation and NF-kappaB-dependent gene expression. TAT-NBD accelerated constitutive PMN apoptosis dose dependently and abrogated LPS-delayed apoptosis. These results provide a proof of principle for peptide delivery by TAT-derived protein transduction domains to specifically inhibit NF-kappaB activity in PMNs. This strategy may help in controlling various cellular functions even in short-lived, transfection-resistant primary human cells

NB1 mediates surface expression of the ANCA antigen proteinase 3 on human neutrophils

Antineutrophil cytoplasmic antibodies (ANCAs) with specificity for proteinase 3 (PR3) are central to a form of ANCA-associated vasculitis. Membrane PR3 (mPR3) is expressed only on a subset of neutrophils. The aim of this study was to determine the mechanism of PR3 surface expression on human neutrophils. Neutrophils were isolated from patients and healthy controls, and hematopoietic stem cells from cord blood served as a model of neutrophil differentiation. Surface expression was analyzed by flow cytometry and confocal microscopy, and proteins were analyzed by Western blot experiments. Neutrophil subsets were separated by magnetic cell sorting. Transfection experiments were carried out in HEK293 and HL60 cell lines. Using neutrophils from healthy donors, patients with vasculitis, and neutrophilic differentiated stem cells we found that mPR3 display was restricted to cells expressing neutrophil glycoprotein NB1, a glycosylphosphatidylinositol (GPI)-linked surface receptor. mPR3 expression was decreased by enzymatic removal of GPI anchors from cell membranes and was absent in a patient with paroxysmal nocturnal hemoglobinuria. PR3 and NB1 coimmunoprecipitated from and colocalized on the neutrophil plasma membrane. Transfection with NB1 resulted in specific PR3 surface binding in different cell types. We conclude that PR3 membrane expression on neutrophils is mediated by the NB1 receptor

Vascular smooth muscle and nitric oxide synthase

ABSTRACT The concept of endothelium-derived relaxing factor (EDRF) put forward in 1980 by Furchgott and Zawadzki implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium diffuses to the underlying vascular smooth muscle, where it modulates vascular tone as well as vascular smooth muscle cell (VSMC) proliferation by increasing cGMP formation with subsequent activation of cGMP-dependent protein kinase. According to this concept, VSMC do not express NOS by themselves. This attractive, simple scheme is now under considerable debate. To address this issue, we designed this study with the use of a novel supersensitive immunocytochemical technique of signal amplification with tyramide and electron microscopic immunogold labeling complemented with Western blotting, as in our recent studies demonstrating NOS in the myocardial and skeletal muscles. We provide the first evidence that, in contrast to the currently accepted view, VSMC in various blood vessels express all three NOS isoforms depending on the blood vessel type. These findings suggest an alternative mechanism by which local NOS expression may modulate vascular functions in an endothelium-independent manner.

Prorenin and renin-induced extracellular signal-regulated kinase 1/2 activation in monocytes is not blocked by aliskiren or the handle-region peptide

The recently cloned (pro)renin receptor [(P)RR] mediates renin-stimulated cellular effects by activating mitogen-activated protein kinases and promotes nonproteolytic prorenin activation. In vivo, (P)RR is said to be blocked with a peptide consisting of 10 amino acids from the prorenin prosegment called the "handle-region" peptide (HRP). We tested whether human prorenin and renin induce extracellular signal-regulated kinase (ERK) 1/2 activation and whether the direct renin inhibitor aliskiren or the HRP inhibits the receptor. We detected the (P)RR mRNA and protein in isolated human monocytes and in U937 monocytes. In U937 cells, we found that both human renin and prorenin induced a long-lasting ERK 1/2 phosphorylation despite angiotensin II type 1 and 2 receptor blockade. In contrast to angiotensin II-ERK signaling, renin and prorenin signaling did not involv

Endogenous angiotensinergic system in neurons of rat and human trigeminal ganglia.

To clarify the role of Angiotensin II (Ang II) in the sensory system and especially in the trigeminal ganglia, we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of Ang II and substance P in the rat and human trigeminal ganglia. The rat trigeminal ganglia expressed substantial amounts of Ang-N- and ACE mRNA as determined by quantitative real time PCR. Renin mRNA was untraceable in rat samples. Cathepsin D was detected in the rat trigeminal ganglia indicating the possibility of existence of pathways alternative to renin for Ang I formation. In situ hybridization in rat trigeminal ganglia revealed expression of Ang-N mRNA in the cytoplasm of numerous neurons. By using immunocytochemistry, a number of neurons and their processes in both the rat and human trigeminal ganglia were stained for Ang II. Post in situ hybridization immunocytochemistry reveals that in the rat trigeminal ganglia some, but not all Ang-N mRNA-positive neurons marked for Ang II. In some neurons Substance P was found colocalized with Ang II. Angiotensins from rat trigeminal ganglia were quantitated by radioimmunoassay with and without prior separation by high performance liquid chromatography. Immunoreactive angiotensin II (ir-Ang II) was consistently present and the sum of true Ang II (1-8) octapeptide and its specifically measured metabolites were found to account for it. Radioimmunological and immunocytochemical evidence of ir-Ang II in neuronal tissue is compatible with Ang II as a neurotransmitter. In conclusion, these results suggest that Ang II could be produced locally in the neurons of rat trigeminal ganglia. The localization and colocalization of neuronal Ang II with Substance P in the trigeminal ganglia neurons may be the basis for a participation and function of Ang II in the regulation of nociception and migraine pathology