Neuronally produced versican V2 renders C-fiber nociceptors IB4 -positive.

A subpopulation of nociceptors, the glial cell line-derived neurotrophic factor (GDNF)-dependent, non-peptidergic C-fibers, expresses a cell-surface glycoconjugate that can be selectively labeled with isolectin B4 (IB4 ), a homotetrameric plant lectin from Griffonia simplicifolia. We show that versican is an IB4 -binding molecule in rat dorsal root ganglion neurons. Using reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization and immunofluorescence experiments on rat lumbar dorsal root ganglion, we provide the first demonstration that versican is produced by neurons. In addition, by probing Western blots with splice variant-specific antibodies we show that the IB4 -binding versican contains only the glycosaminoglycan alpha domain. Our data support V2 as the versican isoform that renders this subpopulation of nociceptors IB4 -positive (+). A subset of nociceptors, the GDNF-dependent non-peptidergic C-fibers can be characterized by its reactivity for isolectin B4 (IB4), a plant lectin from Griffonia simplicifolia. We have previously demonstrated that versican V2 binds IB4 in a Ca2 + -dependent manner. However, given that versican is thought to be the product of glial cells, it was questionable whether versican V2 can be accountable for the IB4-reactivity of this subset of nociceptors. The results presented here prove - for the first time - a neuronal origin of versican and suggest that versican V2 is the molecule that renders GDNF-dependent non-peptidergic C-fibers IB4-positive.

Long-term indoor gunshot exposure of special police forces induces bronchitic reactions and elevated blood lead levels-The Berlin shooting range study.

BACKGROUND: Gunshot emissions contain toxic elements that can harm those frequently exposed, such as police officers. Several years ago, police indoor firing ranges were closed by the Berlin municipality in response to police officer health complaints, and an investigation was launched into the possible respiratory health risks of frequent gunshot emission exposure. We, therefore, conducted an exploratory cross-sectional study to investigate clinical and functional parameters of respiratory health as well as the burden of trace elements in policemen with long-term high exposure to indoor gunshot emissions, compared to low-exposure and control groups. METHODS: We conducted lung function tests and collected blood and urine samples from Berlin police officers and government employees who were divided into three subject groups based on exposure to gunshot emissions: high exposure (n = 53), low exposure (n = 94) and no exposure (n = 76). Lung function was examined using body plethysmography. Blood and urine samples were tested via inductively coupled plasma mass spectrometry for the presence of common gunshot powder elements (antimony, lead and manganese). Exposure and symptoms were assessed using records as well as questionnaires. RESULTS: Higher exposure was associated with more respiratory symptoms during gun shooting practice (64% vs. 21%, P < 0.001) compared to the low-exposure group. Headache, cough, discoloured mucous and shortness of breath were also more common as were some other symptoms. The cough symptomatology of the high-exposure group also persisted significantly longer (median: 0.67 vs. 0.01 days, range: 0 to 5 days, P = 0.029) compared to the low-exposure group. They also showed a lower forced expiratory volume in 1 s/forced vital capacity quotient (Tiffeneau index), P = 0.018 between the three groups and P = 0.005 for the high-exposure group, a possible marker of early, subclinical bronchial obstruction. We observed increased blood lead concentrations depending on subject's age (+1.2% per year, 95% confidence interval: 0.5-1.9%, P < 0.001) and cumulative gunshot exposure (+0.34% per 100 000 shots, 0.02-0.66%, P = 0.037). CONCLUSIONS: These first results suggest that long-term exposure to indoor gunshot emissions induces bronchitic reactions due to repeated irritation of the airways. Higher levels of exposure lead to more negatively impacted lung function and higher blood lead levels with the possible reason that more frequent exposure may mean shorter regeneration phases for the respiratory mucous membrane. We recommend a reduction of exposure to gunshot emissions in order to decrease symptoms and avoid any-even small-deterioration in spirometry.

A functional P2X7 splice variant with an alternative transmembrane domain 1 escapes gene inactivation in P2X7 knock-out mice.

The ATP-activated P2X7 receptor channel is involved in immune function and inflammatory pain and represents an important drug target. Here we describe a new P2X7 splice variant (P2X7(k)), containing an alternative intracellular N terminus and first transmembrane domain encoded by a novel exon 1 in the rodent P2rx7 gene. Whole cell patch clamp recordings of the rat isoform expressed in HEK293 cells revealed an 8-fold higher sensitivity to the agonist Bz-ATP and much slower deactivation kinetics when compared with the P2X7(a) receptor. Permeability measurements in Xenopus oocytes show a high permeability for N-methyl-D-glucamine immediately upon activation, suggesting that the P2X7(k) channel is constitutively dilated upon opening. The rates of agonist-induced dye uptake and membrane blebbing in HEK cells were also increased. PCR analyses and biochemical analysis by SDS-PAGE and BN-PAGE indicate that the P2X7(k) variant escapes gene deletion in one of the available P2X7(-/-) mice strains and is strongly expressed in the spleen. Taken together, we describe a novel P2X7 isoform with distinct functional properties that contributes to the diversity of P2X7 receptor signaling. Its presence in one of the P2X7(-/-) strains has important implications for our understanding of the role of this receptor in health and disease.

Dual expression of mouse and rat VRL-1 in the dorsal root ganglion derived cell line F-11 and biochemical analysis of VRL-1 after heterologous expression.

The vanilloid-like TRP-channel VRL-1 (TRPV2) is a nonselective cation channel expressed by primary sensory neurons and non-neuronal tissues [Caterina, M.J., Rosen, T.A., Tominaga, M., Brake, A.J and Julius, D. (1999) Nature 398, 436-441]. It is one of the six members of the vanilloid-like TRP-channel family which is now termed the TRPV family [Montell, G., Birnbaumer, L., Flockerzi, V., Bindels, R.J., Brutford, E.A., Caterina, M.J., Clapham, D.E., Harteneck, C., Heller, S., Julius, D., Kojima, I., Mori, Y., Penner, R., Prawitt, D., Scharenberg, A.M., Schultz, G., Shimizu, N. and Zhu, M.X. (2002) Mol. Cell 2, 229-231]. As it is a temperature-gated channel, VRL-1 appears to be functionally related to VR1. In contrast to VR1, VRL-1 is activated at a higher temperature threshold and it does not respond to capsaicin or protons. Here we describe the expression of VRL-1 in the rat dorsal root ganglion-derived cell line F-11, a hybridoma of mouse neuroblastoma (N18TG2) and rat dorsal root ganglion cells. We found by RT-PCR that F-11 cells express not only the rat VRL-1, but also its mouse orthologue in a single cell. The F-11 parental cell line N18TG2 also expressed murine VRL-1. Due to its neuronal character, the DRG-derived F-11 cell line provides an experimental system for the study of VRL-1 biochemistry. However, one has to be aware that both the mouse and the rat protein are expressed simultaneously. Furthermore we cloned VRL-1 from rat brain and analyzed its glycosylation and localization in comparison to the endogenously expressed protein in F-11 cells. In contrast to the endogenous VRL-1 the overexpressed protein is glycosylated. Similar to VR1 the glycosylation is N-linked as shown by an deglycosylation assay. Immunofluorescence analysis of the endogenous VRL-1 in F-11 cells gives only weak signals in the cytoplasm whereas the overexpressed rat VRL-1 appears mainly at the plasma membrane.