Interleukin-17A is involved in mechanical hyperalgesia but not in the severity of murine antigen-induced arthritis.

Interleukin-17A (IL-17A) is considered an important pro-inflammatory cytokine but its importance in joint diseases such as rheumatoid arthritis (RA) is unclear. It has also been reported that IL-17A may induce pain but it is unclear whether pro-inflammatory and pro-nociceptive effects are linked. Here we studied in wild type (WT) and IL-17A knockout (IL-17AKO) mice inflammation and hyperalgesia in antigen-induced arthritis (AIA). We found that the severity and time course of AIA were indistinguishable in WT and IL-17AKO mice. Furthermore, the reduction of inflammation by sympathectomy, usually observed in WT mice, was preserved in IL-17AKO mice. Both findings suggest that IL-17A is redundant in AIA pathology. However, in the course of AIA IL-17AKO mice showed less mechanical hyperalgesia than WT mice indicating that IL-17A contributes to pain even if it is not crucial for arthritis pathology. In support for a role of IL-17A and other members of the IL-17 family in the generation of pain we found that sensory neurones in the dorsal root ganglia (DRG) express all IL-17 receptor subtypes. Furthermore, in isolated DRG neurones most IL-17 isoforms increased tetrodotoxin- (TTX-) resistant sodium currents which indicate a role of IL-17 members in inflammation-evoked sensitization of sensory nociceptive neurones.

Neuronal prostaglandin E2 receptor subtype EP3 mediates antinociception during inflammation.

The pain mediator prostaglandin E2 (PGE2) sensitizes nociceptive pathways through EP2 and EP4 receptors, which are coupled to Gs proteins and increase cAMP. However, PGE2 also activates EP3 receptors, and the major signaling pathway of the EP3 receptor splice variants uses inhibition of cAMP synthesis via Gi proteins. This opposite effect raises the intriguing question of whether the Gi-protein-coupled EP3 receptor may counteract the EP2 and EP4 receptor-mediated pronociceptive effects of PGE2. We found extensive localization of the EP3 receptor in primary sensory neurons and the spinal cord. The selective activation of the EP3 receptor at these sites did not sensitize nociceptive neurons in healthy animals. In contrast, it produced profound analgesia and reduced responses of peripheral and spinal nociceptive neurons to noxious stimuli but only when the joint was inflamed. In isolated dorsal root ganglion neurons, EP3 receptor activation counteracted the sensitizing effect of PGE2, and stimulation of excitatory EP receptors promoted the expression of membrane-associated inhibitory EP3 receptor. We propose, therefore, that the EP3 receptor provides endogenous pain control and that selective activation of EP3 receptors may be a unique approach to reverse inflammatory pain. Importantly, we identified the EP3 receptor in the joint nerves of patients with painful osteoarthritis.

Interleukin-17 sensitizes joint nociceptors to mechanical stimuli and contributes to arthritic pain through neuronal interleukin-17 receptors in rodents.

OBJECTIVE: Interleukin-17 (IL-17) is considered a proinflammatory cytokine, but whether neuronal IL-17 receptors contribute to the generation of arthritic pain is unknown. This study was undertaken to explore whether IL-17A acts on neurons, whether it sensitizes joint nociceptors, and whether neutralization of IL-17 is antinociceptive. METHODS: We recorded action potentials from rat joint nociceptors after intraarticular injection of IL-17A. We studied the expression of the IL-17A receptor in the rat dorsal root ganglia (DRG), explored the effect of IL-17A on signaling pathways in cultured rat DRG neurons, and using patch clamp recordings, monitored changes of excitability by IL-17A. We tested whether an antibody to IL-17 influences pain behaviors in mice with antigen-induced arthritis (AIA). RESULTS: A single injection of IL-17A into the rat knee joint elicited a slowly developing and long-lasting sensitization of nociceptive C fibers of the joint to mechanical stimuli, which was not attenuated by neutralizing tumor necrosis factor α or IL-6. The IL-17A receptor was visualized in most rat DRG neurons, the cell bodies of primary sensory neurons. In isolated and cultured rat DRG neurons, IL-17A caused rapid phosphorylation of protein kinase B and ERK, and it rapidly enhanced excitability. In mice with unilateral AIA in the knee, an antibody against IL-17 improved the guarding score and reduced secondary mechanical hyperalgesia at the ipsilateral paw. CONCLUSION: Our findings indicate that IL-17A has the potential to act as a pain mediator by targeting IL-17 receptors in nociceptive neurons, and these receptors are particularly involved in inflammation-evoked mechanical hyperalgesia.

Update on peripheral mechanisms of pain: beyond prostaglandins and cytokines.

The peripheral nociceptor is an important target of pain therapy because many pathological conditions such as inflammation excite and sensitize peripheral nociceptors. Numerous ion channels and receptors for inflammatory mediators were identified in nociceptors that are involved in neuronal excitation and sensitization, and new targets, beyond prostaglandins and cytokines, emerged for pain therapy. This review addresses mechanisms of nociception and focuses on molecules that are currently favored as new targets in drug development or that are already targeted by new compounds at the stage of clinical trials--namely the transient receptor potential V1 receptor, nerve growth factor, and voltage-gated sodium channels--or both.

Effects of prostaglandin D2 on tetrodotoxin-resistant Na+ currents in DRG neurons of adult rat.

Tetrodotoxin-resistant (TTX-R) Na(+) channels play a key role in the generation of action potentials in nociceptive dorsal root ganglion (DRG) neurons and are an important target for the proinflammatory mediator prostaglandin E(2), which augments these currents. Prostaglandin D(2) (PGD(2)) is released in the tissue together with prostaglandin E(2), and it was reported to be antiinflammatory, but its effect on primary afferent neurons is unclear. In the present study we localised G(s)-protein-coupled DP1 and G(i)-protein-coupled DP2 receptors in DRG neurons, and we assessed the effect of PGD(2) on TTX-R Na(+) currents in patch-clamp recordings from small- to medium-sized cultured DRG neurons from adult rats. DP1 and DP2 receptor-like immunoreactivity was localised in the vast majority of DRG neurons. In all neurons, PGD(2) shifted conductance to more hyperpolarised potentials, depending on an action at Na(v)1.9 channels. In about one third of the neurons, PGD(2) additionally influenced Na(v)1.8 channels by facilitating conductance and by increasing maximal current amplitudes. Selective DP1 receptor activation increased the amplitude of TTX-R Na(+) currents of most neurons, but this effect was counteracted by DP2 receptor activation, which by itself had no effect. In the current-clamp mode, PGD(2) lowered the threshold for elicitation of an action potential and increased the number of action potentials per stimulus, an effect mainly depending on DP1 receptor activation. Thus, the net effect of PGD(2) on DRG neurons is pronociceptive, although the magnitude of the TTX-R Na(+) currents depends on the balance of DP1 and DP2 receptor activation.

Tumor necrosis factor causes persistent sensitization of joint nociceptors to mechanical stimuli in rats.

OBJECTIVE: During inflammation in the joint, normal joint movements are usually painful. A neuronal mechanism for this form of mechanical hyperalgesia is the persistent sensitization of joint nociceptors to mechanical stimuli. Because tumor necrosis factor (TNF) is a major mediator of joint inflammation, we undertook the present study both to explore the potential of TNF to sensitize joint nociceptors to mechanical stimuli and to address the cellular mechanism involved. METHODS: In anesthetized rats, action potentials (APs) were recorded from sensory nociceptive Aδ fibers and C fibers supplying the knee joint. We monitored responses to rotation of the knee joint at innocuous and noxious intensities. TNF, etanercept, and a p38 inhibitor were injected into the knee joint, and the cyclooxygenase (COX) inhibitor diclofenac was administered intraperitoneally. APs were also recorded in isolated cultured dorsal root ganglion (DRG) neurons in order to test for changes in neuronal excitability induced by TNF. RESULTS: A single application of TNF into the normal knee joint caused a significant persistent sensitization of nociceptive sensory fibers to mechanical stimuli applied to the joint. This effect was dose dependent. It was prevented by coadministration of etanercept or by an inhibitor of p38, and it was attenuated by systemic application of a COX inhibitor. Patch clamp recordings from isolated DRG neurons showed a rapid increase in neuronal excitability induced by TNF. CONCLUSION: TNF can induce a long-lasting sensitization of joint nociceptors to mechanical stimuli and thus can induce long-lasting mechanical hyperalgesia in joints. TNF can act directly on neurons, underscoring its role as a sensitizing pain mediator.

Blockade of TTX-resistant and TTX-sensitive Na+ currents in cultured dorsal root ganglion neurons by fomocaine and the fomocaine derivative Oe 9000.

Fomocaine and its new derivative Oe 9000 are local anesthetics in which the inner aromatic moiety carries a phenoxymethyl substituent and is linked to the tertiary amine by an alkylene chain, rendering these compounds considerably lipophilic and increasing their chemical and metabolic stability. Although fomocaine was used for surface anesthesia, the presumed mode of action of fomocaine and Oe 9000, the blockade of voltage-gated Na(+) currents in neurons, has not been investigated. In the present experiments we used the whole-cell mode of the patch-clamp technique and studied the effect of both drugs on voltage-gated Na(+) currents in isolated and cultured dorsal root ganglion (DRG) neurons from adult rats. Both drugs reversibly reduced slowly activating and inactivating tetrodotoxin-resistant (TTX-R) Na(+) currents as well as rapidly activating and inactivating TTX-sensitive (TTX-S) Na(+) currents at low micromolar concentrations. For the reduction of TTX-R Na(+) currents the IC(50) of fomocaine was 10.3µM, and the IC(50) for the more hydrophilic Oe 9000 was 4.5µM. These IC(50) values are more than one order of magnitude lower than the corresponding IC(50) of other local anesthetics such as lidocaine. Similar as for other local anesthetics, the effects showed a frequency dependence indicating that the compounds preferentially bind to the open and/or inactivated state of the channel. These data establish for the first time the functional suppression of TTX-R and TTX-S Na(+) currents by fomocaine and Oe 9000 in neurons. They support the further research into the use of Oe 9000 as a novel local anesthetic.

Joint pain.

Both inflammatory and degenerative diseases of joints are major causes of chronic pain. This overview addresses the clinical problem of joint pain, the nociceptive system of the joint, the mechanisms of peripheral and central sensitization during joint inflammation and long term changes during chronic joint inflammation. While the nature of inflammatory pain is obvious the nature and site of origin of osteoarthritic pain is less clear. However, in both pathological conditions mechanical hyperalgesia is the major pain problem, and indeed, both joint nociceptors and spinal nociceptive neurons with joint input show pronounced sensitization for mechanical stimulation. Molecular mechanisms of mechanical sensitization of joint nociceptors are addressed with an emphasis on cytokines, and molecular mechanisms of central sensitization include data on the role of excitatory amino acids, neuropeptides and spinal prostaglandins. The overview will also address long-term changes of pain-related behavior, response properties of neurons and receptor expression in chronic animal models of arthritis.

Functional consequences of leucine and tyrosine mutations in the dual pore motifs of the yeast K(+) channel, Tok1p.

Tandem pore-loop potassium channels differ from the majority of K(+) channels in that a single polypeptide chain carries two K(+)-specific segments (P) each sandwiched between two transmembrane helices (M) to form an MP(1)M-MP(2)M series. Two of these peptide molecules assemble to form one functional potassium channel, which is expected to have biaxial symmetry (commonly described as asymmetric) due to independent mutation in the two MPM units. The resulting intrinsic asymmetry is exaggerated in fungal 2P channels, especially in Tok1p of Saccharomyces, by the N-terminal presence of four more transmembrane helices. Functional implications of such structural asymmetry have been investigated via mutagenesis of residues (L290 in P(1) and Y424 in P(2)) that are believed to provide the outermost ring of carbonyl oxygen atoms for coordination with potassium ions. Both complementary mutations (L290Y and Y424L) yield functional potassium channels having quasi-normal conductance when expressed in Saccharomyces itself, but the P(1) mutation (only) accelerates channel opening about threefold in response to depolarizing voltage shifts. The more pronounced effect at P(1) than at P(2) appears paradoxical in relation to evolution, because a comparison of fungal Tok1p sequences (from 28 ascomycetes) shows the filter sequence of P(2) (overwhelmingly TIGYGD) to be much stabler than that of P(1) (mostly TIGLGD). Profound functional asymmetry is revealed by the fact that combining mutations (L290Y + Y424L)-which inverts the order of residues from the wild-type channel-reduces the expressed channel conductance by a large factor (20-fold, cf.

Calcitonin gene-related peptide enhances TTX-resistant sodium currents in cultured dorsal root ganglion neurons from adult rats.

The neuropeptide calcitonin gene-related peptide (CGRP) binds to a subpopulation of dorsal root ganglion (DRG) neurons, elevates intracellular calcium, and causes inward currents in about 30% of lumbar DRG neurons. Using whole-cell patch clamp recordings, we found in the present study that application of CGRP to isolated and cultured DRG neurons from the adult rat enhances voltage-gated TTX-resistant (TTX-R) Na(+) inward currents in about 30% of small- to medium-sized DRG neurons. During CGRP, peak densities of Na(+) currents increased significantly. CGRP shifted the membrane conductance of the CGRP-responsive cells towards hyperpolarization without changing the slope of the peak conductance curve. The effect of CGRP was blocked by coadministration of CGRP8-37, an antagonist at the CGRP receptor. The effect of CGRP was also blocked after bath application of PKA14-22, a membrane-permeant blocker of protein kinase A, and PKC19-31, a PKC inhibitor, in the recording pipette. These data show pronounced facilitatory effects of CGRP on TTX-R Na(+) currents in DRG neurons which are mediated through CGRP receptors and intracellular pathways involving protein kinases A and C. Thus, in addition to prostaglandins, CGRP is another mediator that affects TTX-R Na(+) currents which are thought to occur mainly in nociceptive DRG neurons.

Plant K(in) and K(out) channels: approaching the trait of opposite rectification by analyzing more than 250 KAT1-SKOR chimeras.

Members of the Shaker-like plant K(+) channel family share a common structure, but are highly diverse in their function: they behave as either hyperpolarization-activated inward-rectifying (K(in)) channels, or leak-like (K(weak)) channels, or depolarization-activated outward-rectifying (K(out)) channels. Here we created 256 chimeras between the K(in) channel KAT1 and the K(out) channel SKOR. The chimeras were screened in a potassium-uptake deficient yeast strain to identify those, which mediate potassium inward currents, i.e., which are functionally equivalent to KAT1. This strategy allowed us to identify three chimeras which differ from KAT1 in three parts of the polypeptide: the cytosolic N-terminus, the cytosolic C-terminus, and the putative voltage-sensor S4. Additionally, mutations in the K(out) channel SKOR were generated in order to localize molecular entities underlying its depolarization activation. The triple mutant SKOR-D312N-M313L-I314G, carrying amino-acid changes in the S6 segment, was identified as a channel which did not display any rectification in the tested voltage-range.

Changes in the effect of spinal prostaglandin E2 during inflammation: prostaglandin E (EP1-EP4) receptors in spinal nociceptive processing of input from the normal or inflamed knee joint.

Inflammatory pain is caused by sensitization of peripheral and central nociceptive neurons. Prostaglandins substantially contribute to neuronal sensitization at both sites. Prostaglandin E2 (PGE2) applied to the spinal cord causes neuronal hyperexcitability similar to peripheral inflammation. Because PGE2 can act through EP1-EP4 receptors, we addressed the role of these receptors in the spinal cord on the development of spinal hyperexcitability. Recordings were made from nociceptive dorsal horn neurons with main input from the knee joint, and responses of the neurons to noxious and innocuous stimulation of the knee, ankle, and paw were studied after spinal application of recently developed specific EP1-EP4 receptor agonists. Under normal conditions, spinal application of agonists at EP1, EP2, and EP4 receptors induced spinal hyperexcitability similar to PGE2. Interestingly, the effect of spinal EP receptor activation changed during joint inflammation. When the knee joint had been inflamed 7-11 hr before the recordings, only activation of the EP1 receptor caused additional facilitation, whereas spinal application of EP2 and EP4 receptor agonists had no effect. Additionally, an EP3alpha receptor agonist reduced responses to mechanical stimulation. The latter also attenuated spinal hyperexcitability induced by spinal PGE2. In isolated DRG neurons, the EP3alpha agonist reduced the facilitatory effect of PGE2 on TTX-resistant sodium currents. Thus pronociceptive effects of spinal PGE2 can be limited, particularly under inflammatory conditions, through activation of an inhibitory splice variant of the EP3 receptor. The latter might be an interesting target for controlling spinal hyperexcitability in inflammatory pain states.