Nociceptive Cell Research Articles

A disintegrin and metalloproteinase domain 17-epidermal growth factor receptor signaling contributes to oral cancer pain.

Cancer cells secrete pronociceptive mediators that sensitize adjacent sensory neurons and cause pain. Identification and characterization of these mediators could pinpoint novel targets for cancer pain treatment. In this study, we identified candidate genes in cancer cell lines that encode for secreted or cell surface proteins that may drive nociception. To undertake this work, we used an acute cancer pain mouse model, transcriptomic analysis of publicly available human tumor-derived cell line data, and a literature review. Cancer cell line supernatants were assigned a phenotype based on evoked nociceptive behavior in an acute cancer pain mouse model. We compared gene expression data from nociceptive and nonnociceptive cell lines. Our analyses revealed differentially expressed genes and pathways; many of the identified genes were not previously associated with cancer pain signaling. Epidermal growth factor receptor (EGFR) and disintegrin metalloprotease domain 17 (ADAM17) were identified as potential targets among the differentially expressed genes. We found that the nociceptive cell lines contained significantly more ADAM17 protein in the cell culture supernatant compared to nonnociceptive cell lines. Cytoplasmic EGFR was present in almost all (>90%) tongue primary afferent neurons in mice. Monoclonal antibody against EGFR, cetuximab, inhibited cell line supernatant-induced nociceptive behavior in an acute oral cancer pain mouse model. We infer from these data that ADAM17-EGFR signaling is involved in cancer mediator-induced nociception. The differentially expressed genes and their secreted protein products may serve as candidate therapeutic targets for oral cancer pain and warrant further evaluation.

(354) Repurposing Druggable Targets for the Treatment of Cancer Pain: A Translational Approach

Cancer cells secrete pro-nociceptive mediators into the microenvironment that sensitize sensory neurons and cause pain. We sought to identify protein encoding genes that drive nociception. We used a preclinical mouse model, transcriptomic analysis, and a proteomic literature review to undertake this work. Cancer cell line supernatant was assigned a phenotype based on evoked nociceptive behavior in a cancer pain mouse model. Mice received 50μl injections of supernatant in the tongue for 3 days followed by orofacial behavior assessment with a dolognawmeter 1 hour after the third injection. Data from the NCBI Gene Expression Omnibus was used to performdifferential gene expression and pathway analysis. We identified proteins potentially responsible for nociception using a literature review of the nociceptive cell lines. Mediator targets produced by these analyses were validated with the cancer pain mouse model. We identified 4 nociceptive and 4 non-nociceptive cell lines. We subsequently identified 1760 differentially expressed genes (DEG) and 14 significantly perturbed pathways by comparing the nociceptive and non-nociceptive cell lines. Thirty of 1760 DEG were reported in the literature to yield over-expressed proteins secreted or anchored to the plasma membrane in at least one of the nociceptive cell lines tested. For these 30 DEG, epidermal growth factor receptor (EGFR) was the most commonly cited druggable target (Drug–Gene Product Interaction Database). To determine if EGFR signaling is involved in cancer supernatant-induced nociception, we administered either EGFR antagonist, Cetuximab, or saline followed by nociceptive cell line supernatant injected into the mouse tongue. Mice that received saline exhibited significantly longer gnaw-time following injection of cancer supernatant (48.8±4.5%) compared to mice that received Cetuximab (13.8±11.8%). DEG and their secreted protein products may serve as therapeutic targets for cancer pain and warrant further evaluation.

Amygdala-prefrontal pathways and the dopamine system affect nociceptive responses in the prefrontal cortex

BackgroundWe previously demonstrated nociceptive discharges to be evoked by mechanical noxious stimulation in the prefrontal cortex (PFC). The nociceptive responses recorded in the PFC are conceivably involved in the affective rather than the sensory-discriminative dimension of pain. The PFC receives dense projection from the limbic system. Monosynaptic projections from the basolateral nucleus of the amygdala (BLA) to the PFC are known to produce long-lasting synaptic plasticity. We examined effects of high frequency stimulation (HFS) delivered to the BLA on nociceptive responses in the rat PFC.ResultsHFS induced long lasting suppression (LLS) of the specific high threshold responses of nociceptive neurons in the PFC. Microinjection of N-methyl-D-aspartic acid (NMDA) receptor antagonists (2-amino-5-phosphonovaleric acid (APV), dizocilpine (MK-801)) and also metabotropic glutamate receptor (mGluR) group antagonists (α-methyl-4-carboxyphenylglycine (MCPG), and 2-[(1S,2S)-2-carboxycyclopropyl]-3-(9H-xanthen-9-yl)-D-alanine (LY341495)), prevented the induction of LLS of nociceptive responses. We also examined modulatory effects of dopamine (DA) on the LLS of nociceptive responses. With depletion of DA in response to 6-hydroxydopamine (6-OHDA) injection into the ipsilateral forebrain bundle, LLS of nociceptive responses was decreased, while nociceptive responses were normally evoked. Antagonists of DA receptor subtypes D2 (sulpiride) and D4 (3-{[4-(4-chlorophenyl) piperazin-1-yl] methyl}-1H-pyrrolo [2, 3-b] pyridine (L-745,870)), microinjected into the PFC, inhibited LLS of nociceptive responses.ConclusionsOur results indicate that BLA-PFC pathways inhibited PFC nociceptive cell activities and that the DA system modifies the BLA-PFC regulatory function.

The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons

NKCC1 is highly expressed in dorsal root ganglion neurons, where it is involved in gating sensory information. In a recent study, it was shown that peripheral nerve injury results in increased NKCC1 activity, not due to an increase in cotransporter expression, but to increased phosphorylation of the cotransporter (Pieraut, S., Matha, V., Sar, C., Hubert, T., Méchaly, I., Hilaire, C., Mersel, M., Delpire, E., Valmier, J., and Scamps, F. (2007) J. Neurosci. 27, 6751-6759). Our laboratory has also identified two Ste20-like kinases that bind and phosphorylate NKCC1: Ste20-related proline-alanine-rich kinase (SPAK) and oxidative-stress response 1 (OSR1). In this study, we show that both kinases are expressed at similar expression levels in spinal cord and dorsal root ganglion neurons, and that both kinases participate equally in the regulation of NKCC1. Using a novel fluorescence method to assay NKCC1 activity in single cells, we show a 50% reduction in NKCC1 activity in DRG neurons isolated from SPAK knockout mice, indicating that another kinase, e.g. OSR1, is present to phosphorylate and activate the cotransporter. Using a nociceptive dorsal root ganglion sensory neuronal cell line, which expresses the same cation-chloride cotransporters and kinases as native DRG neurons, and gene silencing via short hairpin RNA, we demonstrate a direct relationship between kinase expression and cotransporter activity. We show that inactivation of either kinase significantly affects NKCC1 activity, whereas inactivation of both kinases results in an additive effect. In summary, our study demonstrates redundancy of kinases in the regulation of NKCC1 in dorsal root ganglion neurons.

Characterization and function of TWIK-related acid sensing K + channels in a rat nociceptive cell

We examined the properties of a proton sensitive current in acutely dissociated, capsaicin insensitive nociceptive neurons from rat dorsal root ganglion (DRG). The current had features consistent with K + leak currents of the KCNK family (TASK-1, TASK-3; TWIK-related acid sensing K +). Acidity and alkalinity induced inward and outward shifts in the holding current accompanied by increased and decreased whole cell resistance consistent with a K + current. We used alkaline solutions to open the channel and examine its properties. Alkaline evoked currents (AECs; pH 10.0–10.75), reversed near the K + equilibrium potential (−74 mV), and were suppressed 85% in 0 mM K +. AECs were insensitive to Cs + (1 mM) and anandamide (1 μM), but blocked by Ba ++ (1 mM), quinidine (100 μM) or Ruthenium Red (10 μM). This pharmacology was identical to that of rat TASK-3 and inconsistent with that of TASK-1 or TASK-2. The TASK-like AEC was not modulated by PKA (forskolin, κ opioid agonists U69593 and GR8696, somatostatin) but was inhibited by PKC activator phorbol-12-myristate-13 acetate (PMA). When acidic solutions were used, we were able to isolate a Ba ++ and Ruthenium Red insensitive current that was inhibited by Zn ++. This Zn ++ sensitive component of the proton sensitive current was consistent with TASK-1. In current clamp studies, acidic pH produced sensitive changes in resting membrane potential but did not influence excitability (pH 7.2–6.8). In contrast, Zn ++ produced substantial changes in excitability at physiological pH. Alkaline solutions produced hyperpolarization followed by proportional burst discharges (pH 10.75–11.5) and increased excitability (at pH 7.4). In conclusion, multiple TASK currents were present in a DRG nociceptor and differentially contributed to distinct discharge mechanisms.

Different determinants on growth and synapse formation in cultured neurons.

The influence of substrate and target on growth and synapse formation was investigated in identified leech neurons in culture. Nociceptive neurons and annulus erector motoneurons were cultured on ganglion capsules, a substrate they encounter in the leech. Within a few hours, single nociceptive cells sprouted profusely, whereas annulus erector cells failed to grow. When annulus erector neurons were plated near a nociceptive cell on the same capsule both neurons grew, made contact and formed an electrical synapse, different from the chemical synapse they form in the ganglion, but identical to that formed when plated on Concanavalin A. These results suggest that substrate and target have complementary effects regulating growth, but fail to define the type of synapse formed.

What we have learned from the study of learning in the leech

The use of invertebrate preparations has contributed greatly to our understanding of the neural basis of learning. The leech is especially useful for studying behavioral changes and their underlying neuronal mechanisms. Learning in the leech is essentially identical to that found in other animals, both vertebrate and invertebrate. Using anatomical and physiological techniques on leeches as they learn, we have begun to characterize the properties of individual neurons and neuronal networks that play a role in learning. We have been able to show two neuronal mechanisms that have not been previously associated with associative conditioning. The first has to do with the importance of contingency: one stimulus [the conditional stimulus (CS)] becomes associated with a second stimulus [the unconditional stimulus, (US)] in proportion to the ability of the CS to predict the US. We have found that important properties for encoding predictability, such as circuit reconfiguration, may lie in the US pathway. The firing of the serotonergic Retzius cells is taken as the US; consistent CS prediction of a US prevents "dropout" of a critical component of one US pathway. Throughout training, predicted USs continue to elicit a barrage of action potentials in these cells. Recurring unpredicted USs degrade both the learning and the response of the Retzius cell to the US. A second insight is that at least two US pathways contribute to learning, the Retzius cell pathway and the nociceptive (N) cell pathway. This second pathway persists after the elimination of the Retzius cell pathway. The observation of multiple US pathways raises a host of issues concerning CS-US convergence and the functional significance of distinct US pathways, and our results are discussed in terms of implications to current models of learning.

The effects of procaine, strychnine and penicillin on nociceptive neurons in leech segmental ganglia

1. 1. Procaine, strychnine and penicillin selectively depolarized the membrane potential and prolonged the action potential recorded in the lateral but not the medial nociceptive (N) cell in the hiruclinid leech Macrobdella decora. 2. 2. In contrast, procaine did not differentiate between medial and lateral N cells in two other hiruclinid leeches Hirudo medicinalis and Haemopis marmorata. In these species, the drug equally decreased the amplitude of action potentials in both types of N cells without effecting their resting membrane properties. 3. 3. In the nociceptive neurons of the glossiphoniid leech Haementeria ghilianii which possesses only one type of N cell, procaine produced a depolarization and prolonged the action potential. This finding indicates that the single pair of N cells in Haementeria is of the lateral type. 4. 4. The results suggest that the lateral type N cell in Macrobdella and Haementeria share a unique Na +-dependent conductance which is selectively opened by the local anesthetics procaine and strychnine as well as by penicillin. This conductance is either not present or insensitive to the drugs in the homologous N cells in the two other leech species examined.

A monosynaptic connection between the medial nociceptive and the Nut cell in leech ganglia

1. In leech (Macrobdella decora) segmental ganglia, stimulation of the medial nociceptive cell (Nmed) evoked a short latency (2–3 ms) excitatory postsynaptic potential (epsp) of 1–4 mV amplitude in the Nut cell, a nearby identified neuron of unknown function. 2. The epsp's were abolished by high concentrations of Mg and enhanced by high concentrations of Ca. 3. Following intracellular injection of tetraethylammonium chloride (TEA) into the presynaptic Nmed cell, the amplitude of the epsp increased to 5–7 mV in a graded manner. 4. In contrast, no excitatory synaptic responses were obtained in the Nut cell after stimulation of the lateral nociceptive cell (Nlat). 5. These results strongly suggest the presence of a monosynaptic connection between the Nmed and the Nut cell and present more evidence that the two nociceptive cells are not identical.

Cross-reactivities of monoclonal antibodies between select leech neuronal and epithelial tissues.

Three monoclonal antibodies, originally studied because of their neuron-specific staining in the leech central nervous system, are characterized further here, both immunocytochemically and biochemically, with Western blot staining using the central nervous system and peripheral tissues. The three antibodies react both with neurons and select epithelial tissue in the central nervous system, gut, and penis. Antibody Lan3-8 reacts with neurons in the nerve cord and gut but with epithelial cells in the penis; it binds to a 65K molecule in all three tissues. Lan3-2 and Laz2-369 are considered as a related pair because in the central nervous system the former stains all (four) and the latter generally only half (two) of the neurons in a standard midbody ganglion responding to nociceptive stimulation. In the gut, both antibodies label patches of epithelial cells and Laz2-369 stains a previously unknown type of gut neuron. While a given antibody stains different bands in gut and central nervous system immunoblots, comparing the bands of both antibodies for the same tissue extract makes it apparent that there are similarities in the molecular species that both antibodies recognize. For each monoclonal antibody, the histologically identified tissue antigens need to be correlated with proteins identified on Western blots. Of particular interest are the broad 130K bands to which Lan3-2 and Laz2-369 bind. The question is raised whether the molecular species in these bands represent a family of proteins that serve a specific nociceptive cell function.