Combinations of classical and non-classical voltage dependent potassium channel openers suppress nociceptor discharge and reverse chronic pain signs in a rat model of Gulf War illness.

In a companion paper we examined whether combinations of Kv7 channel openers (Retigabine and Diclofenac; RET, DIC) could be effective modifiers of deep tissue nociceptor activity; and whether such combinations could then be optimized for use as safe analgesics for pain-like signs that developed in a rat model of GWI (Gulf War Illness) pain. In the present report, we examined the combinations of Retigabine/Meclofenamate (RET/MEC) and Meclofenamate/Diclofenac (MEC/DIC). Voltage clamp experiments were performed on deep tissue nociceptors isolated from rat DRG (dorsal root ganglion). In voltage clamp studies, a stepped voltage protocol was applied (-55 to -40 mV; Vh=-60 mV; 1500 msec) and Kv7 evoked currents were subsequently isolated by Linopirdine subtraction. MEC greatly enhanced voltage dependent conductance and produced exceptional maximum sustained currents of 6.01 ± 0.26 pA/pF (EC50: 62.2 ± 8.99 µM). Combinations of RET/MEC, and MEC/DIC substantially amplified resting currents at low concentrations. MEC/DIC also greatly improved voltage dependent conductance. In current clamp experiments, a cholinergic challenge test (Oxotremorine-M, 10 µM; OXO), associated with our GWI rat model, produced powerful action potential (AP) bursts (85 APs). Optimized combinations of RET/MEC (5 and 0.5 µM) and MEC/DIC (0.5 and 2.5 µM) significantly reduced AP discharges to 3 and 7 Aps, respectively. Treatment of pain-like ambulatory behavior in our rat model with a RET/MEC combination (5 and 0.5 mg/kg) successfully rescued ambulation deficits, but could not be fully separated from the effect of RET alone. Further development of this approach is recommended.

Development of KVO treatment strategies for chronic pain in a rat model of Gulf War Illness.

We examined whether combinations of Kv7 channel openers could be effective modifiers of deep tissue nociceptor activity; and whether such combinations could then be optimized for use as safe analgesics for pain-like signs that developed in a rat model of GWI (Gulf War Illness) pain. Voltage clamp experiments were performed on subclassified nociceptors isolated from rat DRG (dorsal root ganglion). A stepped voltage protocol was applied (-55 to -40 mV; Vh = -60 mV; 1500 ms) and Kv7 evoked currents were subsequently isolated by linopirdine subtraction. Directly activated and voltage activated K+ currents were characterized in the presence and absence of Retigabine (5-100 µM) and/or Diclofenac (50-140 µM). Retigabine produced substantial voltage dependent effects and a maximal sustained current of 1.14 pA/pF ± 0.15 (ED50: 62.7 ± 3.18 µM). Diclofenac produced weak voltage dependent effects but a similar maximum sustained current of 1.01 ± 0.26 pA/pF (ED50: 93.2 ± 8.99 µM). Combinations of Retigabine and Diclofenac substantially amplified resting currents but had little effect on voltage dependence. Using a cholinergic challenge test (Oxotremorine, 10 µM) associated with our GWI rat model, combinations of Retigabine (5 uM) and Diclofenac (2.5, 20 and 50 µM) substantially reduced or totally abrogated action potential discharge to the cholinergic challenge. When combinations of Retigabine and Diclofenac were used to relieve pain-signs in our rat model of GWI, only those combinations associated with serious subacute side effects could relieve pain-like behaviors.

Behavioral, cellular and molecular maladaptations covary with exposure to pyridostigmine bromide in a rat model of gulf war illness pain.

Many veterans of Operation Desert Storm (ODS) struggle with the chronic pain of Gulf War Illness (GWI). Exposure to insecticides and pyridostigmine bromide (PB) have been implicated in the etiology of this multisymptom disease. We examined the influence of 3 (DEET (N,N-diethyl-meta-toluamide), permethrin, chlorpyrifos) or 4 GW agents (DEET, permethrin, chlorpyrifos, pyridostigmine bromide (PB)) on the post-exposure ambulatory and resting behaviors of rats. In three independent studies, rats that were exposed to all 4 agents consistently developed both immediate and delayed ambulatory deficits that persisted at least 16 weeks after exposures had ceased. Rats exposed to a 3 agent protocol (PB excluded) did not develop any ambulatory deficits. Cellular and molecular studies on nociceptors harvested from 16WP (weeks post-exposure) rats indicated that vascular nociceptor Nav1.9 mediated currents were chronically potentiated following the 4 agent protocol but not following the 3 agent protocol. Muscarinic linkages to muscle nociceptor TRPA1 were also potentiated in the 4 agent but not the 3 agent, PB excluded, protocol. Although Kv7 activity changes diverged from the behavioral data, a Kv7 opener, retigabine, transiently reversed ambulation deficits. We concluded that PB played a critical role in the development of pain-like signs in a GWI rat model and that shifts in Nav1.9 and TRPA1 activity were critical to the expression of these pain behaviors.

DEET potentiates the development and persistence of anticholinesterase dependent chronic pain signs in a rat model of Gulf War Illness pain.

Exposure to DEET (N,N-diethyl-meta-toluamide) may have influenced the pattern of symptoms observed in soldiers with GWI (Gulf War Illness; Haley and Kurt, 1997). We examined how the addition of DEET (400mg/kg; 50% topical) to an exposure protocol of permethrin (2.6mg/kg; topical), chlorpyrifos (CP; 120mg/kg), and pyridostigmine bromide (PB;13mg/kg) altered the emergence and pattern of pain signs in an animal model of GWI pain (Nutter et al., 2015). Rats underwent behavioral testing before, during and after a 4week exposure: 1) hindlimb pressure withdrawal threshold; 2) ambulation (movement distance and rate); and 3) resting duration. Additional studies were conducted to assess the influence of acute DEET (10-100µM) on muscle and vascular nociceptor Kv7, KDR, Nav1.8 and Nav1.9. We report that a 50% concentration of DEET enhanced the development and persistence of pain-signs. Rats exposed to all 4 compounds exhibited ambulation deficits that appeared 5-12weeks post-exposure and persisted through weeks 21-24. Rats exposed to only three agents (CP or PB excluded), did not fully develop ambulation deficits. When PB was excluded, rats also developed rest duration pain signs, in addition to ambulation deficits. There was no evidence that physiological doses of DEET acutely modified nociceptor Kv7, KDR, Nav1.8 or Nav1.9 activities. Nevertheless, DEET augmented protocols decreased the conductance of Kv7 expressed in vascular nociceptors harvested from chronically exposed rats. We concluded that DEET enhanced the development and persistence of pain behaviors, but the anticholinesterases CP and PB played a determinant role.

Exposure to Gulf War Illness chemicals induces functional muscarinic receptor maladaptations in muscle nociceptors.

Chronic pain is a component of the multisymptom disease known as Gulf War Illness (GWI). There is evidence that pain symptoms could have been a consequence of prolonged and/or excessive exposure to anticholinesterases and other GW chemicals. We previously reported that rats exposed, for 8 weeks, to a mixture of anticholinesterases (pyridostigmine bromide, chlorpyrifos) and a Nav (voltage activated Na(+) channel) deactivation-inhibiting pyrethroid, permethrin, exhibited a behavior pattern that was consistent with a delayed myalgia. This myalgia-like behavior was accompanied by persistent changes to Kv (voltage activated K(+)) channel physiology in muscle nociceptors (Kv7, KDR). In the present study, we examined how exposure to the above agents altered the reactivity of Kv channels to a muscarinic receptor (mAChR) agonist (oxotremorine-M). Comparisons between muscle nociceptors harvested from vehicle and GW chemical-exposed rats revealed that mAChR suppression of Kv7 activity was enhanced in exposed rats. Yet in these same muscle nociceptors, a Stromatoxin-insensitive component of the KDR (voltage activated delayed rectifier K(+) channel) exhibited decreased sensitivity to activation of mAChR. We have previously shown that a unique mAChR-induced depolarization and burst discharge (MDBD) was exaggerated in muscle nociceptors of rats exposed to GW chemicals. We now provide evidence that both muscle and vascular nociceptors of naïve rats exhibit MDBD. Examination of the molecular basis of the MDBD in naïve animals revealed that while the mAChR depolarization was independent of Kv7, the action potential burst was modulated by Kv7 status. mAChR depolarizations were shown to be dependent, in part, on TRPA1. We argue that dysfunction of the MDBD could be a functional convergence point for maladapted ion channels and receptors consequent to exposure to GW chemicals.

A delayed chronic pain like condition with decreased Kv channel activity in a rat model of Gulf War Illness pain syndrome.

Following their return from deployment, Gulf War (GW) veterans reported widespread joint and muscle pain at rates that far exceeded those of soldiers returning from other conflicts. It is widely believed that exposure to insecticides, repellants and nerve gas prophylactics contributed to the symptoms of Gulf War Illness (GWI), but an animal model of GW pain has been elusive. In our previous work, we observed that 4-8 weeks exposure to pyridostigmine bromide (PB), permethrin and chlorpyrifos could produce persistent alterations in the physiology of Nav1.9 and Kv7 expressed in deep tissue nociceptors of the dorsal root ganglion. However, behavioral assessments from these same rats were not consistent with a delayed pain syndrome similar to that of GWI pain. In the present studies, we intensified the exposure to anticholinesterases PB and chlorpyrifos while retaining the same dosages. Animals receiving the intensified protocol for 30 days exhibited significant increases in resting for about 8 weeks after exposure. Thereafter, all measures were comparable to controls. Animals treated with intensified anticholinesterases for 60 days exhibited increased resting and reduced movement 12 weeks post-exposure. In whole cell patch studies, muscle and vascular nociceptor KDR and Kv7 ion channels exhibited increased amplitude relative to controls (e.g., normalized current and/or peak conductance) at 8 weeks post-exposures; however, at 12 weeks post-exposure, the amplitude of these currents was significantly decreased in muscle nociceptors. In current clamp studies, muscle nociceptors also manifested increased action potential duration, afterhyperpolarization and increased discharge to muscarinic agonists 12 weeks post-exposure. The decline in activity of muscle nociceptor KDR and Kv7 channel proteins was consistent with increased nociceptor excitability and a delayed myalgia in rats exposed to GW chemicals.

Persistent Na+ and K+ channel dysfunctions after chronic exposure to insecticides and pyridostigmine bromide.

Many soldiers that served in the 1991 Gulf War developed widespread chronic pain. Exposure to insecticides and the nerve gas prophylactic pyridostigmine bromide (PB) was identified as risk factors by the Research Advisory Committee on Gulf War Veterans' Illnesses (GWI). We examined whether a 60 day exposure to neurotoxicants/PB (NTPB) produced behavioral, molecular and cellular indices of chronic pain in the rat. Male rats were exposed to chlorpyrifos (120mg/kg; SC), permethrin (2.6mg/kg; topical), and PB (13.0mg/kg; oral) or their respective vehicles (corn oil, ethanol, and water). Permethrin can exert profound influences on voltage activated Na(+) channel proteins; while chlorpyrifos and PB can increase absorption and/or retard metabolism of permethrin as well as inhibit cholinesterases. During and after exposure to these agents, we assessed muscle pressure pain thresholds and activity (distance and rest time). Eight and 12 weeks after treatments ceased, we used whole cell patch electrophysiology to examine the physiology of tissue specific DRG nociceptor channel proteins expressed in muscle and putative vascular nociceptors (voltage dependent, activation, inactivation, and deactivation). Behavioral indices were unchanged after treatment with NTPB. Eight weeks after treatments ended, the peak and average conductance of Kv7 mediated K(+) currents were significantly increased in vascular nociceptors. When a specific Kv7 inhibitor was applied (linopirdine, 10µM) NTPB treated vascular nociceptors emitted significantly more spontaneous APs than vehicle treated neurons. Changes to Kv7 channel physiology were resolved 12 weeks after treatment. The molecular alterations to Kv7 channel proteins and the specific susceptibility of the vascular nociceptor population could be important for the etiology of GWI pain.

Heterogeneity of nicotinic receptor class and subunit mRNA expression among individual parasympathetic neurons from rat intracardiac ganglia.

Neurons have the potential to form thousands of distinct neuronal nicotinic receptors from the eight alpha and three beta subunits that currently are known. In an effort to determine how much of this potential complexity is realized among individual neurons, we examined the nicotinic pharmacological and biophysical properties and receptor subunit mRNA expression patterns in individual neurons cultured from rat epicardial ganglia. Analysis of the whole-cell pharmacology of these neurons showed a diversity of responses to the agonists acetylcholine, nicotine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium, suggesting that a heterogeneous population of nicotinic receptor classes, or subtypes, is expressed by individual neurons. Single-channel analysis demonstrated three distinct conductances (18, 24, and 31 pS), with patches from different neurons containing different combinations of these channel classes. We used single-cell RT-PCR to examine nicotinic acetylcholine receptor (nAChR) subunit mRNA expression by individual neurons. Although mRNAs encoding all eight neuronal nAChR subunits for which we probed (alpha 2-alpha 5, alpha 7, beta 2-beta 4) were present in multicellular cultures, we found that individual epicardial neurons express distinct subsets of these nAChR subunit mRNAs. These results suggest that individual epicardial neurons express distinct arrays of nAChR subunits and that these subunits may assemble into functional receptors with distinct and variable subunit composition. This variable receptor subunit expression provides an explanation for the diversity of pharmacological and single-channel responses we have observed in individual neurons.

Monovalent and divalent cation permeability and block of neuronal nicotinic receptor channels in rat parasympathetic ganglia.

Acetylcholine-evoked currents mediated by activation of nicotinic receptors in rat parasympathetic neurons were examined using whole-cell voltage clamp. The relative permeability of the neuronal nicotinic acetylcholine (nACh) receptor channel to monovalent and divalent inorganic and organic cations was determined from reversal potential measurements. The channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Cs+ > K+ > Rb+ > Na+ > Li+, and permeability ratios relative to Na+ (Px/PNa) ranging from 1.27 to 0.75. The selectivity of the alkaline earths was also weak, with the sequence of Mg2+ > Sr2+ > Ba2+ > Ca2+, and relative permeabilities of 1.10 to 0.65. The relative Ca2+ permeability (PCa/PNa) of the neuronal nACh receptor channel is approximately fivefold higher than that of the motor endplate channel (Adams, D. J., T. M. Dwyer, and B. Hille. 1980. Journal of General Physiology. 75:493-510). The transition metal cation, Mn2+ was permeant (Px/PNa = 0.67), whereas Ni2+, Zn2+, and Cd2+ blocked ACh-evoked currents with half-maximal inhibition (IC50) occurring at approximately 500 microM, 5 microM and 1 mM, respectively. In contrast to the muscle endplate AChR channel, that at least 56 organic cations which are permeable to (Dwyer et al., 1980), the majority of organic cations tested were found to completely inhibit ACh-evoked currents in rat parasympathetic neurons. Concentration-response curves for guanidinium, ethylammonium, diethanolammonium and arginine inhibition of ACh-evoked currents yielded IC50's of approximately 2.5-6.0 mM. The organic cations, hydrazinium, methylammonium, ethanolammonium and Tris, were measureably permeant, and permeability ratios varied inversely with the molecular size of the cation. Modeling suggests that the pore has a minimum diameter of 7.6 A. Thus, there are substantial differences in ion permeation and block between the nACh receptor channels of mammalian parasympathetic neurons and amphibian skeletal muscle which represent functional consequences of differences in the primary structure of the subunits of the ACh receptor channel.

Calcium permeability and modulation of nicotinic acetylcholine receptor-channels in rat parasympathetic neurons.

Neuronal nicotinic acetylcholine (ACh)-activated currents in rat parasympathetic ganglion cells were examined using whole-cell and single-channel patch clamp recording techniques. The whole-cell current-voltage (I-V) relationship exhibited strong inward rectification and a reversal (zero current) potential of -3.9 mV in nearly symmetrical Na+ solutions (external 140 mM Na+/internal 160 mM Na+). Isosmotic replacement of extracellular Na+ with either Ca2+ or Mg2+ yielded the permeability (Px/PNa) sequence Mg2+ (1.1) > Na+ (1.0) > Ca2+ (0.65). Whole-cell ACh-induced current amplitude decreased as [Ca2+]0 was raised from 2.5 mM to 20 mM, and remained constant at higher [Ca2+]0. Unitary ACh-activated currents recorded in excised outside-out patches had conductances ranging from 15-35 pS with at least three distinct conductance levels (33 pS, 26 pS, 19 pS) observed in most patches. The neuronal nicotinic ACh receptor-channel had a slope conductance of 30 pS in Na+ external solution, which decreased to 20 pS in isotonic Ca2+ and was unchanged by isosmotic replacement of Na+ with Mg2+. ACh-activated single channel currents had an apparent mean open time (tau 0) of 1.15 +/- 0.16 ms and a mean burst length (tau b) of 6.83 +/- 1.76 ms at -60 mV in Na+ external solution. Ca(2+)-free external solutions, or raising [Ca2+]0 to 50-100 mM decreased both the tau 0 and tau b of the nAChR channel. Varying [Ca2+]0 produced a marked decrease in NP0, while substitution of Mg2+ for Na+ increased NP0. These data suggest that activation of the neuronal nAChR channel permits a substantial Ca2+ influx which may modulate Ca(2+)-dependent ion channels and second messenger pathways to affect neuronal excitability in parasympathetic ganglia.

Physicochemical characterization of the alpha-peptide of the sodium channel from rat brain.

The alpha-peptide of the rat brain sodium channel of apparent molecular weight 260K has been purified to homogeneity in order to determine its structural and chemical properties. By negative-stain electron microscopy, the molecule morphology of the solubilized channel protein appears as a stack of disks or rouleaux whose dimensions are 40 A X 200 A. Measurement of the secondary structure by circular dichroism shows that the alpha-peptide is a conformationally flexible polypeptide that contains mostly beta-sheet and random-coil in mixed detergent-phospholipid micelles and folds into a conformation that has approximately 65% alpha-helix after reconstitution into phosphatidylcholine vesicles. Preparative polyacrylamide gel electrophoresis was used to obtain a chemically homogeneous peptide to analyze the amino acid and carbohydrate composition. The amino acid composition shows a reasonably high content of acidic amino acids with no striking excess of hydrophobic amino acids, while carbohydrate analyses show that carbohydrate is 31% by weight of the protein with sialic acid representing over 50% of the total carbohydrates. The high alpha-helical content, the amino acid composition, and the large carbohydrate mass are similar to those of the eel electroplax sodium channel and appear to be general features of the sodium channels which have been analyzed structurally and chemically to date.

Functional unit size of the neurotoxin receptors on the voltage-dependent sodium channel.

Radiation inactivation was used in situ to determine the functional unit sizes of the neurotoxin receptors of the voltage-dependent sodium channel from rat brain. Frozen or lyophilized synaptosomes were irradiated with high energy electrons generated by a linear accelerator and assayed for [3H]saxitoxin, 125I-Leiurus quinquestriatus quinquestriatus (alpha-scorpion toxin), 125I-Centruroides suffusus suffusus (beta-scorpion toxin), and batrachotoxinin-A 20 alpha-[3H]benzoate binding activity. The functional unit size of the neurotoxin receptors determined in situ by target analysis are 220,000 for saxitoxin, 263,000 for alpha-scorpion toxin, and 45,000 for beta-scorpion toxin. Analysis of the inactivation curve for batrachotoxinin-A 20 alpha-benzoate binding to the channel yields two target sizes of Mr approximately 287,000 (50%) and approximately 51,000 (50%). The results are independent of the purity of the membrane preparation. Comparison of the radiation inactivation data with the protein composition of the rat brain sodium channel indicates that there are at least two functional components.

Preparation and characterization of fluorescent scorpion toxins from Leiurus quinquestriatus quinquestriatus as probes of the sodium channel of excitable cells.

Fluorescent derivatives of scorpion toxin V from Leiurus quinquestriatus quinquestriatus have been prepared so that the topographical, dynamic, and cellular properties of the neurotoxin receptor site on the voltage-dependent sodium channel could be studied. Four different modification strategies have been pursued in which acylated, amidinylated, thio-amidinylated, and reductively alkylated scorpion toxins were prepared. Acylation induces a loss of net positive charge on the toxin and these derivatives are purified by preparative isoelectric focusing and ion-exchange chromatography. Amidinylation and reductive alkylation preserve the protonation state of the toxin and maintain the native tertiary structure of the toxin. Because the native toxin does not contain cysteine, we have introduced new sulfhydryls through modification with the cyclic imidoester 2-iminothiolane which also preserves the net charge on the toxin. Novel purification methods with small amounts of toxin by immunoprecipitation using antibodies directed against the chromophores or through covalent thiol-disulfide exchange chromatography have been utilized. The biological activities, equilibrium binding, and spectroscopic properties indicate that these derivatives retain high affinity for the sodium channel and are as active or only 2-3 times less active than L. quinquestriatus V toxin itself. The spectroscopic properties of these fluorescent derivatives cover the absorption range from 290 to 470 nm, and fluorescence emissions range from 360 to 550 nm where suitable filters and spectral overlap with previously synthesized fluorescent tetrodotoxin can be found. The fluorescent properties in particular show excellent environmental sensitivity and are suitable for probing the molecular dynamics of the toxin receptor and for topographic mapping of the sodium channel by fluorescence resonance energy transfer measurements.

Mapping the molecular structure of the voltage-dependent sodium channel. Distances between the tetrodotoxin and Leiurus quinquestriatus quinquestriatus scorpion toxin receptors.

The Leiurus quinquestriatus quinquestriatus receptor site of the voltage-dependent sodium channel has been characterized using several fluorescent scorpion toxins. The derivatives show fluorescence enhancements upon binding to the receptor site on the channel together with blue shifts. The fluorescence properties of the bound probes indicate a conformationally flexible, hydrophobic site. Binding of tetrodotoxin has no effect on the fluorescence spectra of the bound derivatives, whereas binding of the allosteric activator batrachotoxin enhances the fluorescence about 2-fold and causes a red shift in the emission spectra, suggesting a batrachotoxin-induced conformational change in the scorpion toxin receptor. The distance between the tetrodotoxin receptor and the Leiurus scorpion toxin receptor on the channel was measured by fluorescence resonance energy transfer. Five different chromophoric scorpion toxin derivatives were used as energy transfer acceptors or donors with anthraniloyltetrodotoxin or N-methylanthraniloylglycine-tetrodotoxin as the energy donor or acceptor. Because of the presence of three tetrodotoxin receptors for each Leiurus receptor, the positions of the donors and acceptors were exchanged. Efficiencies of transfer were measured by both donor quenching and sensitized emission. The average distance of separation between these sites is 35 A. Upon batrachotoxin addition, this distance changes to 42 A indicating a conformational change in one subunit of the channel or a change in the interaction between two subunits coupled to the batrachotoxin-binding site. On the basis of these studies, we present a model suggesting that tetrodotoxin binds to a subunit/site which is extracellularly placed and is 35 A from the Leiurus subunit/site which is located in a protein cleft of the channel which extends partly into the membrane, and undergoes a neurotoxin and voltage-dependent conformational change.