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 tumorinduced heat hyperalgesia and nociceptor sensitization were prevented by systemic treatment with the tumor necrosis factor ␣ (TNF␣) antagonist etanercept. In naive mice, exogenous TNF␣ evoked heat hyperalgesia in vivo and sensitized nociceptive nerve fibers to heat in vitro. TNF␣ 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 TNF␣ as a key player in cancer-related heat hyperalgesia and nociceptor sensitization that generates TRPV1 upregulation and sensitization via TNFR2.

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 Fick's 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.

Ca2+ is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca 2+ concentration ([Ca 2+ ]) c leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage-gated Ca 2+ channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca 2+ entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a "fusion-activated" Ca 2+ 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 Ca 2+ channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X 4 receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X 4 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 P2X 4 . 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 Ca 2+ influx via vesicular Ca 2+ channels regulates fusion pore expansion and vesicle content release.egulated secretion is a fundamental cellular process in many different types of eukaryotic cells, with Ca 2+ -triggered exocytosis being the key element (1-4). Multiple Ca 2+ -dependent steps have been elucidated that ultimately lead to fusion of exocytic vesicles with the plasma membrane, resulting in formation of an aqueous channel, the fusion pore, through which vesicle contents are released (5-8). Although the molecular composition of the fusion pore remains elusive, there is a general acceptance that fusion pores are not merely passive structures, but that their opening and closure are highly regulated and control, or even fine-tune, vesicle content secretion (9-14). Voltage-gated Ca 2+ channels are not present (25). After LB fusion with the plasma membrane, surfactant, a water-insoluble bulky complex, largely remains entrapped within the fused vesicles (26) in which the fusion pores behave as regulated valves or mechanical barriers for release (16,27). As a result, in vitro full content release can be delayed for minutes up to hours (28).We recently reported a "fusion-activated" Ca 2+ entry (FACE) mechanism as a phenomenon in the postfusion phase of surfactant secretion (29). Given that this Ca 2+ signal occasionally spreads throughout the cell, we speculated that it might be important for triggering...

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-␦ via Gab1/2/PI 3 K 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.

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 posttraumatic 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 Ϫ channel inhibitor niflumic acid and by preincubation with the G-protein inhibitor GDP-␤-S. Notably, S1P 3 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 S1P 3 Ϫ/Ϫ mice, whereas in control S1P 1 floxed (S1P 1 fl/fl ) mice and mice with a nociceptor-specific deletion of S1P 1 Ϫ/Ϫ receptor (SNS-S1P 1 Ϫ/Ϫ ), 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-proteindependent activation of an excitatory Cl Ϫ conductance that is largely mediated by S1P 3 receptors present in nociceptors, and point to these receptors as valuable therapeutic targets for post-traumatic pain.