Norbert Mair

Endogenous Tumor Necrosis Factor α (TNFα) Requires TNF Receptor Type 2 to Generate Heat Hyperalgesia in a Mouse Cancer Model

To provide a tool to investigate the mechanisms inducing and maintaining cancer-related pain and hyperalgesia, a soft tissue tumor/metastasis model was developed that is applicable in C57BL/6J wild-type and transgenic mice. We show that the experimental tumor-induced heat hyperalgesia and nociceptor sensitization were prevented by systemic treatment with the tumor necrosis factor α (TNFα) antagonist etanercept. In naive mice, exogenous TNFα evoked heat hyperalgesia in vivo and sensitized nociceptive nerve fibers to heat in vitro. TNFα enhanced the expression of the nociceptor-specific heat transducer ion channel transient receptor potential vanilloid 1 (TRPV1) and increased the amplitudes of capsaicin and heat-activated ionic currents via p38/MAP (mitogen-activated protein) kinase and PKC (protein kinase C). Deletion of the tumor necrosis factor receptor type 2 (TNFR2) gene attenuated heat hyperalgesia and prevented TRPV1 upregulation in tumor-bearing mice, whereas TNFR1 gene deletion played a minor role. We propose endogenous TNFα as a key player in cancer-related heat hyperalgesia and nociceptor sensitization that generates TRPV1 upregulation and sensitization via TNFR2.

A Key Role for gp130 Expressed on Peripheral Sensory Nerves in Pathological Pain

Interleukin-6 (IL-6) is a key mediator of inflammation. Inhibitors of IL-6 or of its signal transducing receptor gp130 constitute a novel class of anti-inflammatory drugs, which raise great hopes for improved treatments of painful inflammatory diseases such as rheumatoid arthritis. IL-6 and gp130 may enhance pain not only indirectly through their proinflammatory actions but also through a direct action on nociceptors (i.e., on neurons activated by painful stimuli). We found indeed that the IL-6/gp130 ligand-receptor complex induced heat hypersensitivity both in vitro and in vivo. This process was mediated by activation of PKC-δ via Gab1/2/PI3K and subsequent regulation of TRPV1, a member of the transient receptor potential (TRP) family of ion channels. To assess the relevance of this direct pain promoting effect of IL-6, we generated conditional knock-out mice, which lack gp130 specifically in nociceptors, and tested them in models of inflammatory and tumor-induced pain. These mice showed significantly reduced levels of inflammatory and tumor-induced pain but no changes in immune reactions or tumor growth. Our results uncover the significance of gp130 expressed in peripheral pain sensing neurons in the pathophysiology of major clinical pain disorders and suggest their use as novel pain relieving agents in inflammatory and tumor pain.

Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579–1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Ficks law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.

Fusion-activated Ca2+ entry via vesicular P2X4 receptors promotes fusion pore opening and exocytotic content release in pneumocytes

Ca2+ is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca2+ concentration ([Ca2+])c leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage-gated Ca2+ channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca2+ entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a “fusion-activated“ Ca2+ entry (FACE) mechanism in alveolar type II (ATII) epithelial cells. FACE follows initial fusion pore opening with a delay of 200–500 ms. The site, molecular mechanisms, and functions of this mechanism remain unknown, however. Here we show that vesicle-associated Ca2+ channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X4 receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X4 receptors. Furthermore, analysis of fluorophore diffusion into and out of individual vesicles after exocytotic fusion provides evidence that FACE regulates postfusion events of LB exocytosis via P2X4. Fusion pore dilation was clearly correlated with the amplitude of FACE, and content release from fused LBs was accelerated in fusions followed by FACE. Based on these findings, we propose a model for regulation of the exocytotic postfusion phase in nonexcitable cells in which Ca2+ influx via vesicular Ca2+ channels regulates fusion pore expansion and vesicle content release.

Threshold calcium levels for lamellar body exocytosis in type II pneumocytes

Pulmonary surfactant is secreted via exocytosis of lamellar bodies (LBs) by alveolar type II cells. Here we analyzed the dependence of LB exocytosis on intracellular Ca2+concentration ([Ca2+]i). In fura 2-loaded cells, [Ca2+]iwas selectively elevated by flash photolysis of a cell-permeant caged Ca2+ compound ( o-nitrophenyl EGTA-AM) or by gradually enhancing cellular Ca2+influx. Simultaneously, surfactant secretion by single cells was analyzed with the fluorescent dye FM 1-43, enabling detection of exocytotic events with a high temporal resolution (T. Haller, J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. Proc. Natl. Acad. Sci. USA 95: 1579-1584, 1998). Exocytosis was initiated at a threshold concentration near 320 nmol/l with both instantaneous or gradual [Ca2+]ielevations. The exocytotic response to flash photolysis was highest during the first minute after the rise in [Ca2+]iand thus almost identical to purinoceptor stimulation by ATP. Correspondingly, the effects of ATP on initial secretion could be sufficiently explained by its ability to mobilize Ca2+. This was further demonstrated by the fact that exocytosis is significantly blocked by suppression of the ATP-induced Ca2+ signal below ∼300 nmol/l. Our results suggest a highly Ca2+-sensitive step in LB exocytosis.

Pulmonary consequences of a deep breath revisited.

About two decades ago, a model was proposed for surfactant release by lung distension. This model implies rapid fusion of lamellar bodies (LBs) with the plasma membrane followed by quick release of surfactant into the alveolus, as reflected by immediate facilitation of lung inflation after a single deep breath. Recent experimental evidence indicates that this two-pool model (intracellular versus alveolar surfactant pool) has to be refined by introducing a third pool, which resides in fused but non-released LBs. Here we discuss the implication of this additional pool for strain-induced surfactant secretion and propose a revised model for the sequence of events following a single deep breath.

Sphingosine-1-phosphate-induced nociceptor excitation and ongoing pain behavior in mice and humans is largely mediated by S1P3 receptor.

The biolipid sphingosine-1-phosphate (S1P) is an essential modulator of innate immunity, cell migration, and wound healing. It is released locally upon acute tissue injury from endothelial cells and activated thrombocytes and, therefore, may give rise to acute post-traumatic pain sensation via a yet elusive molecular mechanism. We have used an interdisciplinary approach to address this question, and we find that intradermal injection of S1P induced significant licking and flinching behavior in wild-type mice and a dose-dependent flare reaction in human skin as a sign of acute activation of nociceptive nerve terminals. Notably, S1P evoked a small excitatory ionic current that resulted in nociceptor depolarization and action potential firing. This ionic current was preserved in “cation-free” solution and blocked by the nonspecific Cl− channel inhibitor niflumic acid and by preincubation with the G-protein inhibitor GDP-β-S. Notably, S1P3 receptor was detected in virtually all neurons in human and mouse DRG. In line with this finding, S1P-induced neuronal responses and spontaneous pain behavior in vivo were substantially reduced in S1P3−/− mice, whereas in control S1P1 floxed (S1P1fl/fl) mice and mice with a nociceptor-specific deletion of S1P1−/− receptor (SNS-S1P1−/−), neither the S1P-induced responses in vitro nor the S1P-evoked pain-like behavior was altered. Therefore, these findings indicate that S1P evokes significant nociception via G-protein-dependent activation of an excitatory Cl− conductance that is largely mediated by S1P3 receptors present in nociceptors, and point to these receptors as valuable therapeutic targets for post-traumatic pain.

Construction of a Global Pain Systems Network Highlights Phospholipid Signaling as a Regulator of Heat Nociception

The ability to perceive noxious stimuli is critical for an animals survival in the face of environmental danger, and thus pain perception is likely to be under stringent evolutionary pressure. Using a neuronal-specific RNAi knock-down strategy in adult Drosophila, we recently completed a genome-wide functional annotation of heat nociception that allowed us to identify α2δ3 as a novel pain gene. Here we report construction of an evolutionary-conserved, system-level, global molecular pain network map. Our systems map is markedly enriched for multiple genes associated with human pain and predicts a plethora of novel candidate pain pathways. One central node of this pain network is phospholipid signaling, which has been implicated before in pain processing. To further investigate the role of phospholipid signaling in mammalian heat pain perception, we analysed the phenotype of PIP5Kα and PI3Kγ mutant mice. Intriguingly, both of these mice exhibit pronounced hypersensitivity to noxious heat and capsaicin-induced pain, which directly mapped through PI3Kγ kinase-dead knock-in mice to PI3Kγ lipid kinase activity. Using single primary sensory neuron recording, PI3Kγ function was mechanistically linked to a negative regulation of TRPV1 channel transduction. Our data provide a systems map for heat nociception and reinforces the extraordinary conservation of molecular mechanisms of nociception across different species.

Oncostatin M induces heat hypersensitivity by gp130-dependent sensitization of TRPV1 in sensory neurons

Oncostatin M (OSM) is a member of the interleukin-6 cytokine family and regulates eg. gene activation, cell survival, proliferation and differentiation. OSM binds to a receptor complex consisting of the ubiquitously expressed signal transducer gp130 and the ligand binding OSM receptor subunit, which is expressed on a specific subset of primary afferent neurons. In the present study, the effect of OSM on heat nociception was investigated in nociceptor-specific gp130 knock-out (SNS-gp130-/- ) and gp130 floxed (gp130fl/fl ) mice.Subcutaneous injection of pathophysiologically relevant concentrations of OSM into the hind-paw of C57BL6Jwild type mice significantly reduced paw withdrawal latencies to heat stimulation. In contrast to gp130fl/fl mice, OSM did not induce heat hypersensitivity in vivo in SNS-gp130-/- mice. OSM applied at the receptive fields of sensory neurons in in vitro skin-nerve preparations showed that OSM significantly increased the discharge rate during a standard ramp-shaped heat stimulus. The capsaicin- and heat-sensitive ion channel TRPV1, expressed on a subpopulation of nociceptive neurons, has been shown to play an important role in inflammation-induced heat hypersensitivity. Stimulation of cultured dorsal root ganglion neurons with OSM resulted in potentiation of capsaicin induced ionic currents. In line with these recordings, mice with a null mutation of the TRPV1 gene did not show any signs of OSM-induced heat hypersensitivity in vivo.The present data suggest that OSM induces thermal hypersensitivity by directly sensitizing nociceptors via OSMR-gp130 receptor mediated potentiation of TRPV1.

Sphingosine 1-phosphate to p38 signaling via S1P1 receptor and Gαi/o evokes augmentation of capsaicin-induced ionic currents in mouse sensory neurons

The perception of painful thermal stimuli by sensory neurons is largely mediated by TRPV1. Upon tissue injury or inflammation, S1P is secreted by thrombocytes as part of an inflammatory cocktail, which sensitizes nociceptive neurons towards thermal stimuli. S1P acts on G-protein coupled receptors that are expressed in sensory neurons and sensitize TRPV1 channels towards thermal stimuli. In this study, the S1P mediated signaling pathway required for sensitization of TRPV1 channels was explored.The capsaicin induced peak inward current (ICAPS) of sensory neurons was significantly increased after S1P stimulation within minutes after application. The potentiation of ICAPS resulted from activation of Gαi through G-protein coupled receptors for S1P. Consequently, Gαi led to a signaling cascade, involving phosphoinositide-3-kinase (PI3K) and protein kinase C, which augmented ICAPS in nociceptive neurons. The S1P1 receptor agonist SEW2871 resulted in activation of the same signaling pathway and potentiation of ICAPS. Furthermore, the mitogen-activated protein kinase p38 was phosphorylated after S1P stimulation and inhibition of p38 signaling by SB203580 prevented the S1P-induced ICAPS potentiation. The current data suggest that S1P sensitized ICAPS through G-protein coupled S1P1 receptor activation of Gαi-PI3K-PKC-p38 signaling pathway in sensory neurons.