Keith R. Bley

The role of IP prostanoid receptors in inflammatory pain

Prostanoid receptor-mediated sensitization of sensory nerve fibres is a key contributor to the generation of hyperalgesia. It is generally thought that prostaglandin (PG) E2 is the principal pro-inflammatory prostanoid. Consequently, prostanoid EP receptors on sensory neurones have been identified as potential therapeutic targets. However, IP prostanoid receptors are also present on sensory neurones, and recent data from transgenic mice lacking the IP receptor demonstrate its importance in the induction of oedema and pain behaviour. PGI2, the primary endogenous agonist for the IP receptor, is rapidly produced following tissue injury or inflammation; thus, it may be of equal, or greater, importance than PGE2 during episodes of inflammatory pain. In this review, Keith Bley, John Hunter, Richard Eglen and Jacqueline Smith compare the roles of EP and IP receptors in nociception and suggest that the IP receptor constitutes a novel target for anti-nociceptive agents.

Tetrodotoxin inhibits neuropathic ectopic activity in neuromas, dorsal root ganglia and dorsal horn neurons

Abstract Neuropathic pain or persistent dysesthesias may be initiated by mechanical, chemical, or ischemic damage to peripheral sensory nerves. In animal models of neuropathic pain, transection or constrictive injury to peripheral nerves produces ectopic discharges originating at both injury sites and related dorsal root ganglia (DRG), and, consequently, hyperexcitability in associated dorsal horn (DH) neurons of the spinal cord. Since ectopic discharges are inhibited by agents that block voltage‐sensitive Na+ channels, it has been postulated that accumulation of Na+ channels in the membrane at nerve injury sites may contribute to, or be responsible for, the development of ectopic neuronal activity (ENA). The present study therefore, tested the sensitivity of ENA to intravenously administered tetrodotoxin (TTX), an extremely potent and selective Na+ channel blocker. Comparative effects of TTX on cardiac parameters such as heart rate (HR) and diastolic blood pressure (DBP) were also studied. Experiments were performed on adult male Sprague‐Dawley rats in which the common sciatic nerve had been transected 4–10 days earlier. Neuromal activity was measured in fine bundles of microfilaments teased from sciatic nerves, and extracellular microelectrode recordings were made from DRG and DH neurons. Cardiovascular parameters were recorded simultaneously. Intravenously administered TTX induced dose‐dependent inhibition of ENA, with that originating from neuromas being the most sensitive; ED50 values (expressed as &mgr;g/kg, with 95% confidence limits) for neuromal, DRG and DH neuron activity were: 0.8 (0.6–1.2), 4.3 (2.2–8.4) and 36.2 (16.1–81.3), respectively. Inhibition of ENA in neuromas and DRG did not recover within 10 min after 100 or 300 &mgr;g/kg TTX. By comparison, the ED50 value for the initial decrease of HR was 17.9 (15.0–21.5) &mgr;g/kg, and partial recovery occurred within ˜3 min. These data support the hypothesis that Na+ channel accumulation contributes to the generation of ectopic discharges in neuromas and DRG, and suggest that TTX‐sensitive Na+ channels located at the nerve injury site and DRG play an important role in the genesis of neuropathic pain.

Reduced heat sensitivity and epidermal nerve fiber immunostaining following single applications of a high-concentration capsaicin patch

Abstract Capsaicin‐containing plant extracts have been used as topical treatments for a variety of pain syndromes for many centuries. Current products containing capsaicin in low concentrations (usually 0.025–0.075% w/w) have shown efficacy against a variety of pain conditions in clinical studies. However, in order to produce significant analgesic effects, these formulations require frequent re‐dosing, often as much as three to five times daily for several weeks. Previous functional and immunohistochemical studies following prolonged exposures to low‐concentration capsaicin cream suggested that the duration and onset of analgesic efficacy correlate with a reduction of cutaneous nociceptive sensory nerve fiber responsiveness and immunostaining. The purpose of the present study was to determine whether a single topical application of a high‐concentration capsaicin‐containing (8% w/w) patch for 120 min or less would induce similar effects on cutaneous nociceptive nerve fibers. Seven days following patch application, changes in heat and cold perception thresholds were determined by quantitative sensory testing and punch biopsies were collected to assess epidermal nerve fiber (ENF) immunostaining density at the application site using PGP 9.5 as a marker. The results show a significant reduction of heat, but not cold, sensitivity and reduction of ENF immunostaining with high‐capsaicin concentration patch applications for 60 or 120 min, compared to placebo patch applications. Application sites exposed to low‐capsaicin concentration (0.04% w/w) patches for 120 min or high‐concentration patches for 30 min were not significantly different from placebo with respect to either thermal threshold detection or ENF immunostaining. The ability of a single 60 min high‐concentration patch application to mimic effects produced by prolonged exposure to low‐concentration capsaicin creams suggests a new approach to the management of chronic pain syndromes.

QX-314 inhibits ectopic nerve activity associated with neuropathic pain

In animal models of neuropathic pain, transection or constrictive injury to peripheral nerves produces ectopic discharges originating at both injury sites and related dorsal root ganglia (DRG). In addition, hyperexcitability is observed in associated dorsal horn (DH) neurons of the spinal cord. As ectopic discharges are inhibited by agents that block voltage-sensitive Na+ channels, it has been postulated that accumulation of Na+ channels in the membrane at nerve injury sites may contribute to the development of ectopic nerve activity (ENA). The goal of the present study was to compare the sensitivity of ENA to lidocaine and QX-314, a positively charged lidocaine derivative, which is frequently assumed to be membrane impermeant. Experiments were performed on adult male Sprague-Dawley rats in which the common sciatic nerve had been transected 4-10 days earlier. Extracellular microelectrode recordings were made from DRG and DH neurons, and neuronal activity was measured in fine bundles of microfilaments teased from sciatic nerves in anesthetized and paralyzed rats. Comparative effects on heart rate (HR) and mean blood pressure (MBP) were also studied. To confirm that externally applied QX-314 is able to inhibit high frequency activity in sensory nerves, QX-314 was superfused over isolated rat vagus nerves during stimulation of compound action potentials in C-fibers (C-spikes). As expected, intravenously administered lidocaine inhibited ENA at all three sites. Lidocaine ED50 values (expressed as mg/kg, with 95% confidence limits) were: 10.2 (7.8-13.3), 1.4 (0.8-2.4) and 0.9 (0.4-2.0) for neuromas, DRG and DH neurons, respectively. QX-314 also induced dose-dependent inhibition of ENA at neuromas and DRG, but produced only a small inhibition of DH neuron ENA. QX-314 had the following ED50 values (mg/kg) for neuromas, DRG and DH neurons, respectively: 2.3 (2.0-2.8), 6.9 (4.7-26.5) and 85.7. QX-314-mediated inhibition of DRG ENA had a slow onset and was long-lasting, relative to lidocaine. Lidocaine or QX-314 also significantly reduced HR and MBP in the same dose range as that which reduced ENA in DRG or neuromas. In isolated rat vagus nerve recordings, QX-314 induced marked use-dependent inhibition of C-spike amplitude, with IC50 values (microM) of 9000 (4600-18,000) and 350 (290-420) for low- (0.03 Hz) and high-frequency (30 Hz) C-spikes, respectively. These data support the hypothesis that Na+ channel accumulation contributes to the generation of ectopic discharges in neuromas and DRG, and suggests that intravenous QX-314 can acutely block Na+ channels at these sites.

Characterization of prostanoid receptor-evoked responses in rat sensory neurones.

Prostanoid receptor‐mediated sensitization, or excitation, of sensory nerve fibres contributes to the generation of hyperalgesia. To characterize the prostanoid receptors present on sensory neurones, biochemical assays were performed on primary cultures of adult rat dorsal root ganglia (DRG) and the F‐11 (embryonic rat DRG×neuroblastoma hybrid) cell line. In DRG cultures, the IP receptor agonists, cicaprost and carbaprostacyclin (cPGI2) stimulated cyclic AMP accumulation. Prostaglandin E2 (PGE2) also increased cyclic AMP levels, but to a lesser extent, while carbocyclic thromboxane A2 (cTxA2), PGD2 and PGF2α had negligible effects. The rank order of agonist potency was cicaprost >PGE2=BMY45778=cPGI2=PGI2. In the F‐11 cells, the rank order of agonist potency for the stimulation of cyclic AMP accumulation was: cicaprost>iloprost=cPGI2=PGI2=BMY45778>PGE2=cTXA2. In DRG cultures, cicaprost induced significantly more accumulation of inositol phosphates than PGE2. To examine the effects of prostanoids on C‐fibre activity, extracellular recordings of d.c. potentials from the rat isolated vagus nerve were made with the ‘grease‐gap’ technique. PGI2 (0.1 nM–10 μM) produced the largest depolarizations of the nerve. The rank order of agonist potency was: PGI2=cPGI2=PGE1>cTXA2>PGE2=PGD2=TXB2>PGF2α. Prior depolarization of nerves with either forskolin (10 μM) or phorbol dibutyrate (1 μM) alone significantly reduced the response to PGI2 (10 μM), while simultaneous application of both forskolin and phorbol dibutyrate attenuated PGI2 responses almost completely. Putative EP1 and/or TP receptor‐selective antagonists had no effect on the responses to PGI2, cPGI2 or PGE2 in the three preparations studied. Collectively, these data are consistent with a positive coupling of IP receptors to both adenylyl cyclase and phospholipase C in sensory neurones. These findings suggest that IP receptors play a major role in the sensitization of rat sensory neurones.

RO1138452 and RO3244794: characterization of structurally distinct, potent and selective IP (prostacyclin) receptor antagonists

Prostacyclin (PGI2) possesses various physiological functions, including modulation of nociception, inflammation and cardiovascular activity. Elucidation of these functions has been hampered by the absence of selective IP receptor antagonists. Two structurally distinct series of IP receptor antagonists have been developed: 4,5‐dihydro‐1H‐imidazol‐2‐yl)‐[4‐(4‐isopropoxy‐benzyl)‐phenyl]‐amine (RO1138452) and R‐3‐(4‐fluoro‐phenyl)‐2‐[5‐(4‐fluoro‐phenyl)
‐benzofuran‐2‐ylmethoxycarbonylamino]‐propionic acid (RO3244794). RO1138452 and RO3244794 display high affinity for IP receptors. In human platelets, the receptor affinities (pKi) were 9.3±0.1 and 7.7±0.03, respectively; in a recombinant IP receptor system, pKi values were 8.7±0.06 and 6.9±0.1, respectively. Functional antagonism of RO1138452 and RO3244794 was studied by measuring inhibition of carbaprostacyclin‐induced cAMP accumulation in CHO‐K1 cells stably expressing the human IP receptor. The antagonist affinities (pKi) of RO1138452 and RO3244794 were 9.0±0.06 and 8.5±0.11, respectively. Selectivity profiles for RO1138452 and RO3244794 were determined via a panel of receptor binding and enzyme assays. RO1138452 displayed affinity at I2 (8.3) and PAF (7.9) receptors, while RO3244794 was highly selective for the IP receptor: pKi values for EP1 (<5), EP3 (5.38), EP4 (5.74) and TP (5.09). RO1138452 (1–10 mg kg−1, i.v.) and RO3244794 (1–30 mg kg−1, i.v.) significantly reduced acetic acid‐induced abdominal constrictions. RO1138452 (3–100 mg kg−1, p.o.) and RO3244794 (0.3–30 mg kg−1, p.o.) significantly reduced carrageenan‐induced mechanical hyperalgesia and edema formation. RO3244794 (1 and 10 mg kg−1, p.o.) also significantly reduced chronic joint discomfort induced by monoiodoacetate. These data suggest that RO1138452 and RO3244794 are potent and selective antagonists for both human and rat IP receptors and that they possess analgesic and anti‐inflammatory potential.

Investigation of the prostacyclin (IP) receptor antagonist RO1138452 on isolated blood vessel and platelet preparations

The current study examined the utility of the recently described prostacyclin (prostanoid IP) receptor antagonist RO1138452 (2‐(4‐(4‐isopropoxybenzyl)‐phenylamino) imidazoline) as a tool for classifying prostanoid receptors.

A stable prostacyclin analog enhances ectopic activity in rat sensory neurons following neuropathic injury

Prostanoids sensitize sensory afferents during inflammation. However, their role in neuropathic pain is still unclear. We analyzed the actions of prostanoids, non-selective (indomethacin) or selective (celecoxib and NS-398) cyclooxygenase-2 (COX or COX-2) inhibitors, on the ectopic activity of dorsal root ganglia (DRG) and dorsal horn (DH) neurons in a model of neuropathic injury. Extracellular recordings of DRG and DH neurons and cardiovascular measurements were performed on anesthetized, paralyzed and artificially ventilated adult male Sprague-Dawley rats whose sciatic nerve had been transected. PGD(2), PGE(2), PGF(2alpha), carbaprostacyclin (cPGI(2); a stable prostacyclin analog), and carbocyclic thromboxane (cTXA(2)) were administered at cumulative doses (0.0001-5 mg/kg, i.p.) at 5 or 10 min intervals. Only cPGI(2) significantly increased the DRG and DH activity in a dose-dependent manner, with ED(50) values of 0.05 (0.01-0.96) and 0.69 (0.11-1.04) mg/kg, respectively. The other prostanoids did not significantly increase activity, although they reduced heart rate for up to 5 min following administration. Time course experiments with single doses of cPGI(2) (1 mg/kg, i.v.) increased DH discharge rate 3-17 min after injection. Indomethacin (3 mg/kg, s.c.), but not celecoxib or NS-398 (both at 6 mg/kg, s.c.), reduced both DRG and DH activity. Our results indicate that cPGI(2) excites DRG and DH neurons of neuropathic rats, and may suggest a role for IP prostanoid receptors in pain episodes associated with nerve injury. The inhibitory effect of indomethacin, but not celecoxib or NS-398, on ectopic activity may suggest that a tonic generation of PGI(2) by COX-1 could contribute to neuropathic pain.