Simultaneous changes of the spontaneous and stimulus-evoked cortical activity in rats acutely treated with mercuric chloride

In earlier studies of our laboratory and in several other reports, alterations in the electrical activity of the cortex of experimental animals on subchronic mercury (Hg) administration were described. In the present work, simultaneous changes in the spontaneous and stimulus-evoked cortical activity elicited by acute administration of inorganic Hg were evaluated with the aim of finding any correlation of the two, possibly giving insight into the mechanism of the alterations. In young adult male Wistar rats, spontaneous cortical, as well as stimulus-evoked cortical and peripheral nervous activity was recorded, before and after acute administration of 3.5 and 7.0 mg/kg Hg2+ ip. The effects of Hg2+ appeared within 10 min and most became significant over 3 h. On the cortex, slowed spontaneous activity, as well as increased amplitude and latency of the evoked potentials (EPs) was seen, and in the periphery, decreased nerve conduction velocity. These alterations seemed to be consistent with a separate cortical and peripheral axonal effect of Hg. Š 2003 Elsevier Inc. All rights reserved

Anti-calmodulins and Tricyclic Adjuvants in Pain Therapy Block the TRPV1 Channel

Ca2+-loaded calmodulin normally inhibits multiple Ca2+-channels upon dangerous elevation of intracellular Ca2+ and protects cells from Ca2+-cytotoxicity, so blocking of calmodulin should theoretically lead to uncontrolled elevation of intracellular Ca2+. Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca2+-uptake via the vanilloid inducible Ca2+-channel/inflamatory pain receptor 1 (TRPV1), which suggests that calmodulin inhibitors may block pore formation and Ca2+ entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced 45Ca2+-uptake at µM concentrations: calmidazolium (broad range)≥trifluoperazine (narrow range)>chlorpromazine/amitriptyline>fluphenazine>>W-7 and W-13 (only partially). Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca2+ or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca2+-uptake in intact TRPV1+ cells, and suggests an extracellular site of inhibition. TRPV1+, inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca2+-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca2+-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca2+-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca2+-channels but not affecting motoneurons

Human keratinocytes are vanilloid resistant

BACKGROUND: Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca(2+)-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. METHODS: To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. RESULTS: Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca(2+)-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1-50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca(2+)-cytotoxicity ([RTX]>15 microM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca(2+)-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. CONCLUSION: TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials

Divalent Heavy Metal Cations Block the TRPV1 Ca(2+) Channel

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel involved in pain sensation and in a wide range of non-pain-related physiological and pathological conditions. The aim of the present study was to explore the effects of selected heavy metal cations on the function of TRPV1. The cations ranked in the following sequence of pore-blocking activity: Co2+ [half-maximal inhibitory concentration (IC50)013 μM]>Cd2+ (IC500 38 μM)>Ni2+ (IC50062 μM)>Cu2+(IC500200 μM). Zn2+ proved to be a weak (IC50027 μM) and only partial inhibitor of the channel function, whereas Mg2+, Mn2+ and La3+ did not exhibit any substantial effect. Co2+, the most potent channel blocker, was able not only to compete with Ca2+ but also to pass with it through the open channel of TRPV1. In response to heat activation or vanilloid treatment, Co2+ accumulation was verified in TRPV1-transfected cell lines and in the TRPV1+ dorsal root ganglion neurons. The inhibitory effect was also demonstrated in vivo. Co2+ applied together with vanilloid agonists attenuated the nocifensive eye wipe response in mice. Different rat TRPV1 pore point mutants (Y627W, N628W, D646N and E651W) were created that can validate the binding site of previously used channel blockers in agonist-evoked 45Ca2+ influx assays in cells expressing TRPV1. The IC50 of Co2+ on these point mutants were determined to be reasonably comparable to those on the wild type, which suggests that divalent cations passing through the TRPV1 channel use the same negatively charged amino acids as Ca2+

The Hero’s Journey: constructing continuity from discontinuity in millennial career changers’ narratives

Although career construction theory is relevant to today's vocational climate, empirical research into it is scarce. Accordingly, we explored this theory by investigating the concepts, structures and processes that UK-based millennial career changers use to construct narratives allowing for continuity of plot and discontinuity of career direction. Interpretative phenomenological analysis on semi-structured interviews (N = 6) identified four themes: dissatisfaction, realisation, sacrifice and return. The Hero’s Journey was identified as an overarching structure for meaning making in career change. Participants assimilated instability and discontinuity into a broader framework of continuity, through narrating a quest for closer alignment between their work and personality. This study provides useful insights into career construction theory and suggests further utility of The Hero’s Journey in career counselling/guidance.N/