The impact of cannabinoids on methemoglobin formation and hemoglobin oxygen affinity: An ex-vivo study.

The affinity of hemoglobin (Hb) to oxygen (O2) influences processes of oxygen delivery and extraction at the tissue level. Despite cannabinoids being utilized or ingested in various ways, their possible impact on Hb-O2 affinity has barely been studied. This is an experimental ex-vivo trial. Venous blood samples were drawn from 5 male and 6 female healthy volunteers and subsequently exposed to different cannabinoid types: (delta-9-tetrahydrocannabinol [Δ9-THC], delta-8-tetrahydrocannabinol [Δ8-THC], cannabidiol [CBD]) at different concentrations. Oxygen dissociation curves (ODC) were measured and blood gas analyses were performed for methemoglobin (MetHb) determination. The results revealed no MetHb formation. Besides two statistically significant changes (+1.4 mmHg and -0.9 mmHg) in the female cohort, following Δ9-THC and Δ8-THC exposure, no further P50 changes could be observed. The study demonstrated an in-vitro effect of selected cannabinoids and dosages on P50 values in female participants, with variations not observed at other dosages, leaving the underlying mechanisms open for debate. MetHb formation, as potential mechanism, was not detected in this study. The precise reasons why changes only occurred at specific dosages remain unclear, indicating a need for further in-vivo research to understand the interaction between cannabinoids and Hb-O2 affinity completely.

The effect of desflurane, isoflurane and sevoflurane on the hemoglobin oxygen dissociation curve in human blood samples.

Desflurane, isoflurane and sevoflurane, three halogenated ethers, are commonly used inhaled anesthetics, both in the operating room and in the intensive care unit (ICU). The potency and dosage of these drugs is expressed by the MAC value (minimum alveolar concentration). Their interaction with hemoglobin and its affinity for oxygen, best described by the oxygen dissociation curve (ODC), has already been investigated, with conflicting results. Altered by many factors, the ODC can be shifted to the left or to the right, therefore increasing or decreasing hemoglobin oxygen (Hb-O2) affinity. In venous blood samples of 22 healthy participants (11 female, 11 male) ODC were measured with a high-throughput method in vitro. Blood samples were either exposed to control or to three different concentrations of desflurane, isoflurane or sevoflurane prior to and during measurements (low, medium and high corresponding to MAC 0.5, MAC 1.0 and MAC 2.0). With increasing concentrations from control to medium, desflurane and isoflurane significantly decreased Hb-O2 affinity by shifting the ODC to the right (p = 0.016 and p < 0.001) but sevoflurane showed no effects. When further increasing concentrations from medium to high, all three inhaled anesthetics shifted the ODC back to the left (p < 0.001). Comparing only controls to high concentrations, a significant increase in Hb-O2 affinity for desflurane (p = 0.005) and sevoflurane (p < 0.001) was detected. Our study shows a varying effect at different doses of inhaled anesthetics on Hb-O2 affinity. While the underlying mechanisms remain unclear, these results show an effect which needs to be further investigated to determine if patients undergoing anesthesia may potentially benefit or get disadvantage from this slightly increased (e.g. impaired pulmonary oxygen uptake), or decreased Hb-O2 affinity (e.g. arterial vascular disease).Trial registration: This study is registered with clinicaltrials.gov (NCT04612270).

The impact of nebulized epoprostenol and iloprost on hemoglobin oxygen affinity: an ex vivo experiment.

Inhalational prostacyclins act as strong vasodilators, potentially improving oxygenation by reducing shunt fraction and ventilation-perfusion mismatch. As prostacyclin receptors are known to be present on human erythrocytes, possible direct effects on hemoglobin oxygen transport were further explored by examining the sole in vitro influence of prostacyclins on hemoglobin oxygen (Hb-O2) affinity. Venous blood samples from 20 healthy volunteers were exposed in vitro to supramaximal doses of epoprostenol, iloprost, and compared with control. By high-throughput measurements, hemoglobin oxygen dissociation curves (ODCs) were derived. Hb-O2 affinity, expressed by P50 and Hill coefficient, was determined and analyzed for three subgroups: males (n = 10), females not taking oral contraceptives (n = 4), and females taking oral contraceptives (n = 6). Epoprostenol significantly decreased P50 in all (males, females without contraceptives, and females taking oral contraceptives) [27.5 (26.4-28.6) mmHg (control) vs. 24.2 (22.7-25.3) mmHg; P < 0.001. median (interquartile range, IQR)] thereby increasing Hb-O2 affinity. Inversely, iloprost only showed significant effects in females taking oral contraceptives where P50 was markedly increased and therefore Hb-O2 affinity decreased [28.4 (27.9-28.9) mmHg (control) vs. 34.4 (32.2-36.0) mmHg; P < 0.001]. Prostacyclin-receptor stimulation and subsequent cAMP-mediated ATP release from erythrocytes are discussed as a possible underlying mechanism for the effect of epoprostenol on Hb-O2 affinity. The reason for the sex hormone-modified iloprost effect remains unclear. Being aware of potentially differing effects on Hb-O2 affinity might help select the right prostacyclin (epoprostenol vs. iloprost) depending on the patient and the underlying disease (e.g., acute respiratory distress syndrome vs. peripheral arterial disease).

Genetic and functional evidence for gp130/IL6ST-induced transient receptor potential ankyrin 1 upregulation in uninjured but not injured neurons in a mouse model of neuropathic pain.

ABSTRACT: Peripheral nerve injuries result in pronounced alterations in dorsal root ganglia, which can lead to the development of neuropathic pain. Although the polymodal mechanosensitive transient receptor potential ankyrin 1 (TRPA1) ion channel is emerging as a relevant target for potential analgesic therapies, preclinical studies do not provide unequivocal mechanistic insight into its relevance for neuropathic pain pathogenesis. By using a transgenic mouse model with a conditional depletion of the interleukin-6 (IL-6) signal transducer gp130 in Nav1.8 expressing neurons (SNS-gp130-/-), we provide a mechanistic regulatory link between IL-6/gp130 and TRPA1 in the spared nerve injury (SNI) model. Spared nerve injury mice developed profound mechanical hypersensitivity as indicated by decreased withdrawal thresholds in the von Frey behavioral test in vivo, as well as a significant increase in mechanosensitivity of unmyelinated nociceptive primary afferents in ex vivo skin-nerve recordings. In contrast to wild type and control gp130fl/fl animals, SNS-gp130-/- mice did not develop mechanical hypersensitivity after SNI and exhibited low levels of Trpa1 mRNA in sensory neurons, which were partially restored by adenoviral gp130 re-expression in vitro. Importantly, uninjured but not injured neurons developed increased responsiveness to the TRPA1 agonist cinnamaldehyde, and neurons derived from SNS-gp130-/- mice after SNI were significantly less responsive to cinnamaldehyde. Our study shows for the first time that TRPA1 upregulation is attributed specifically to uninjured neurons in the SNI model, and this depended on the IL-6 signal transducer gp130. We provide a solution to the enigma of TRPA1 regulation after nerve injury and stress its significance as an important target for neuropathic pain disorders.

Dose- and Sex-Dependent Changes in Hemoglobin Oxygen Affinity by the Micronutrient 5-Hydroxymethylfurfural and α-Ketoglutaric Acid.

5-Hydroxymethylfurfural (5-HMF) is known to increase hemoglobin oxygen affinity (Hb-O2 affinity) and to induce a left shift of the oxygen dissociation curve (ODC). It is under investigation as a therapeutic agent in sickle cell anemia and in conditions where pulmonary oxygen uptake is deteriorated or limited (e.g., various clinical conditions or altitude exposure). The combination of 5-HMF and α-ketoglutaric acid (αKG) is commercially available as a nutritional supplement. To further elucidate dose effects, ODCs were measured in vitro in venous whole blood samples of 20 healthy volunteers (10 female and 10 male) after the addition of three different doses of 5-HMF, αKG and the combination of both. Linear regression analysis revealed a strong dose-dependent increase in Hb-O2 affinity for 5-HMF (R2 = 0.887; p < 0.001) and the commercially available combination with αKG (R2 = 0.882; p < 0.001). αKG alone increased Hb-O2 affinity as well but to a lower extent. Both the combination (5-HMF + αKG) and 5-HMF alone exerted different P50 and Hill coefficient responses overall and between sexes, with more pronounced effects in females. With increasing Hb-O2 affinity, the sigmoidal shape of the ODC was better preserved by the combination of 5-HMF and αKG than by 5-HMF alone. Concerning the therapeutic effects of 5-HMF, this study emphasizes the importance of adequate dosing in various physiological and clinical conditions, where a left-shifted ODC might be beneficial. By preserving the sigmoidal shape of the ODC, the combination of 5-HMF and αKG at low (both sexes) and medium (males only) doses might be able to better maintain efficient oxygen transport, particularly by mitigating potentially deteriorated oxygen unloading in the tissue. However, expanding knowledge on the interaction between 5-HMF and Hb-O2 affinity in vitro necessitates further investigations in vivo to additionally assess pharmacokinetic mechanisms.

High-throughput determination of oxygen dissociation curves in a microplate reader-A novel, quantitative approach.

In vitro determination of the hemoglobin oxygen dissociation curve (ODC) requires highly elaborate, specialized, and costly technical equipment. In addition, there is a lack of methods that combine reliable ODC recordings with high throughput in small blood samples for routine analysis. We here introduce a modified, commercial 96-well plate with an integrated unidirectional gas flow system specifically adapted for use in fluorescence microplate readers. Up to 92 samples of whole or hemolyzed, buffered or unbuffered blood, including appropriate controls or internal standard hemoglobin solutions, can be analyzed within ~25 min. Oxygen saturation is measured in each well with dual wavelength spectroscopy, and oxygen partial pressure using fluorescence lifetime of commercial oxygen sensors at the in- and outlet ports of the gas-flow system. Precision and accuracy of this method have been determined and were compared with those of a standard method. We further present two applications that exemplarily highlight the usefulness and impact of this novel approach for clinical diagnostics or basic research.

Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

TNF-α-converting enzyme, a member of the ADAM (A disintegrin and metalloproteinase) protease family and also known as ADAM17, regulates inflammation and regeneration in health and disease. ADAM17 targets are involved in pain development and hypersensitivity in animal models of inflammatory and neuropathic pain. However, the role of ADAM17 in the pain pathway is largely unknown. Therefore, we used the hypomorphic ADAM17 (ADAM17ex/ex) mouse model to investigate the importance of ADAM17 in nociceptive behavior, morphology, and function of primary afferent nociceptors. ADAM17ex/ex mice were hyposensitive to noxious stimulation, showing elevated mechanical thresholds as well as impaired heat and cold sensitivity. Despite these differences, skin thickness and innervation were comparable to controls. Although dorsal root ganglia of ADAM17ex/ex mice exhibited normal morphology of peptidergic and nonpeptidergic neurons, a small but significant reduction in the number of isolectin ß-4-positive neurons was observed. Functional electrical properties of unmyelinated nociceptors showed differences in resting membrane potential, afterhyperpolarization, and firing patterns in specific subpopulations of sensory neurons in ADAM17ex/ex mice. However, spinal cord morphology and microglia activity in ADAM17ex/ex mice were not altered. Our data suggest that ADAM17 contributes to the processing of painful stimuli, with a complex mode of action orchestrating the function of neurons along the pain pathway.-Quarta, S., Mitric, M., Kalpachidou, T., Mair, N., Schiefermeier-Mach, N., Andratsch, M., Qi, Y., Langeslag, M., Malsch, P., Rose-John, S., Kress, M. Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl- Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in numerous physiological and pathophysiological processes. We have previously reported a S1P-induced nocifensive response in mice by excitation of sensory neurons via activation of an excitatory chloride current. The underlying molecular mechanism for the S1P-induced chloride conductance remains elusive. In the present study, we identified two CLCN voltage-gated chloride channels, CLCN3 and CLCN5, which mediated a S1P-induced excitatory Cl- current in sensory neurons by combining RNA-seq, adenovirus-based gene silencing and whole-cell electrophysiological voltage-clamp recordings. Downregulation of CLCN3 and CLCN5 channels by adenovirus-mediated delivery of shRNA dramatically reduced S1P-induced Cl- current and membrane depolarization in sensory neurons. The mechanism of S1P-induced activation of the chloride current involved Rho GTPase but not Rho-associated protein kinase. Although S1P-induced potentiation of TRPV1-mediated ionic currents also involved Rho-dependent process, the lack of correlation of the S1P-activated Cl- current and the potentiation of TRPV1 by S1P suggests that CLCN3 and CLCN5 are necessary components for S1P-induced excitatory Cl- currents but not for the amplification of TRPV1-mediated currents in sensory neurons. This study provides a novel mechanistic insight into the importance of bioactive sphingolipids in nociception.

Changes in Ionic Conductance Signature of Nociceptive Neurons Underlying Fabry Disease Phenotype.

The first symptom arising in many Fabry patients is neuropathic pain due to changes in small myelinated and unmyelinated fibers in the periphery, which is subsequently followed by a loss of sensory perception. Here we studied changes in the peripheral nervous system of Fabry patients and a Fabry mouse model induced by deletion of α-galactosidase A (Gla-/0). The skin innervation of Gla-/0 mice resembles that of the human Fabry patients. In Fabry diseased humans and Gla-/0 mice, we observed similar sensory abnormalities, which were also observed in nerve fiber recordings in both patients and mice. Electrophysiological recordings of cultured Gla-/0 nociceptors revealed that the conductance of voltage-gated Na+ and Ca2+ currents was decreased in Gla-/0 nociceptors, whereas the activation of voltage-gated K+ currents was at more depolarized potentials. Conclusively, we have observed that reduced sensory perception due to small-fiber degeneration coincides with altered electrophysiological properties of sensory neurons.

Involvement of Peripheral Nerves in the Transgenic PLP-α-Syn Model of Multiple System Atrophy: Extending the Phenotype.

UNLABELLED: Multiple system atrophy (MSA) is a fatal, rapidly progressive neurodegenerative disease with (oligodendro-)glial cytoplasmic α-synuclein (α-syn) inclusions (GCIs). Peripheral neuropathies have been reported in up to 40% of MSA patients, the cause remaining unclear. In a transgenic MSA mouse model featuring GCI-like inclusion pathology based on PLP-promoter driven overexpression of human α-syn in oligodendroglia motor and non-motor deficits are associated with MSA-like neurodegeneration. Since α-syn is also expressed in Schwann cells we aimed to investigate whether peripheral nerves are anatomically and functionally affected in the PLP-α-syn MSA mouse model. RESULTS: To this end, heat/cold as well as mechanical sensitivity tests were performed. Furthermore, in vivo and ex vivo nerve conduction and the G-ratios of the sciatic nerve were analyzed, and thermosensitive ion channel mRNA expression in dorsal root ganglia (DRG) was assessed. The presence of human α-syn in Schwann cells was associated with subtle behavioral impairments. The G-ratio of the sciatic nerve, the conduction velocity of myelinated and unmyelinated primary afferents and the expression of thermosensitive ion channels in the sensory neurons, however, were similar to wildtype mice. CONCLUSION: Our results suggest that the PNS appears to be affected by Schwann cell α-syn deposits in the PLP-α-syn MSA mouse model. However, there was no consistent evidence for functional PNS perturbations resulting from such α-syn aggregates suggesting a more central cause of the observed behavioral abnormalities. Nonetheless, our results do not exclude a causal role of α-syn in the pathogenesis of MSA associated peripheral neuropathy.

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.

Hypoxia-inducible factor 1 regulates heat and cold pain sensitivity and persistence.

AIMS: The present study assessed the functions of the transcription factor hypoxia-inducible factor (HIF) in sensory neurons in models of acute, inflammatory, ischemic, and neuropathic pain. The alpha subunit, HIF1α, was specifically deleted in neurons of the dorsal root ganglia by mating HIF1α(fl/fl) mice with SNScre mice. RESULTS: SNS-HIF1α(-/-) mice were more sensitive to noxious heat and cold pain stimulation than were HIF1α(fl/fl) control mice. They also showed heightened first-phase nociceptive responses in the formalin and capsaicin tests with increased numbers of cFos-positive neurons in the dorsal horn, and intensified hyperalgesia in early phases after paw inflammation and hind limb ischemia/reperfusion. The behavioral cold and heat pain hypersensitivity was explained by increased calcium fluxes after transient receptor potential channel activation in primary sensory neurons of SNS-HIF1α(-/-) mice and lowered electrical activation thresholds of sensory fibers. SNS-HIF1α(-/-) mice however, developed less neuropathic pain after sciatic nerve injury, which was associated with an abrogation of HIF1-mediated gene up-regulation. INNOVATION: The results suggest that HIF1α is protective in terms of acute heat and cold pain but in case of ongoing activation in injured neurons, it may promote the development of neuropathic pain. CONCLUSION: The duality of HIF1 in pain regulation may have an impact on the side effects of drugs targeting HIF1, which are being developed, for example, as anticancer agents. Specifically, in patients with cancer neuropathy, however, temporary HIF1 inhibition might provide a welcome combination of growth and pain reduction.

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-ß-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)(-/-) mice, whereas in control S1P(1) floxed (S1P(1)(fl/fl)) mice and mice with a nociceptor-specific deletion of S1P(1)(-/-) receptor (SNS-S1P(1)(-/-)), 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 S1P(3) 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 animal's 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 C57BL6J wild 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.

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

Ca(2+) 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.

Genetic evidence for involvement of neuronally expressed S1P1 receptor in nociceptor sensitization and inflammatory pain.

Sphingosine-1-phosphate (S1P) is a key regulator of immune response. Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue. However, it is not known if S1P acts on the endings of nociceptive neurons, thereby contributing to the generation of inflammatory pain. We found that the S1P1 receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors. Both S1P and agonists at the S1P1 receptor induced hypersensitivity to noxious thermal stimulation in vitro and in vivo. S1P-induced hypersensitivity was strongly attenuated in mice lacking TRPV1 channels. S1P and inflammation-induced hypersensitivity was significantly reduced in mice with a conditional nociceptor-specific deletion of the S1P1 receptor. Our data show that neuronally expressed S1P1 receptors play a significant role in regulating nociceptor function and that S1P/S1P1 signaling may be a key player in the onset of thermal hypersensitivity and hyperalgesia associated with inflammation.

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-delta 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.

Endogenous tumor necrosis factor alpha (TNFalpha) 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 alpha (TNFalpha) antagonist etanercept. In naive mice, exogenous TNFalpha evoked heat hyperalgesia in vivo and sensitized nociceptive nerve fibers to heat in vitro. TNFalpha 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 TNFalpha as a key player in cancer-related heat hyperalgesia and nociceptor sensitization that generates TRPV1 upregulation and sensitization via TNFR2.

Inhibition by cytoplasmic nucleotides of a new cation channel in freshly isolated human and rat type II pneumocytes.

Here we report a 26- to 29-pS cation channel abundantly expressed in freshly isolated and primary cultured type II cells from rat or healthy human lungs. The channel was never spontaneously active in cell-attached patches but could be activated by cell permeabilization with beta-escin. Excised patch-clamp experiments revealed activation by Ca(2+) concentrations at the cytoplasmic side in the micromolar range. High concentrations of amiloride (>10 microM) at the extracellular side did not inhibit. The channel was equally permeable for K(+) and Na(+) but was essentially impermeable for Cl(-), Ca(2+), and Mg(2+). It was blocked by adenosine nucleotides (cytoplasmic side) with the following order of potency: AMP approximately ADP (EC(50) ATP >> adenosine >> cyclic AMP. The blocking effect of ATP was reproduced by its nonhydrolyzable analogs AMPPNP or ATP-gamma-S. GTP did not inhibit. Cd(2+) blocked the channel with an EC(50) approximately 55.5 nM. We conclude that type II cells express a Ca(2+)-dependent, nucleotide-inhibited, nonselective, and Ca(2+)-impermeable cation channel (NSC(Ca/AMP)) with tonically suppressed activity. RT-PCR confirmed expression of TRPM4b, a channel with functional characteristics almost identical with NSC(Ca/AMP). Potential physiological roles are discussed.