Alla Khodorova

Endothelin-B receptor activation triggers an endogenous analgesic cascade at sites of peripheral injury

Endothelin-1 (ET-1) is a newly described pain mediator that is involved in the pathogenesis of pain states ranging from trauma to cancer. ET-1 is synthesized by keratinocytes in normal skin and is locally released after cutaneous injury. While it is able to trigger pain through its actions on endothelin-A (ETA) receptors of local nociceptors, it can coincidentally produce analgesia through endothelin-B (ETB) receptors. Here we map a new endogenous analgesic circuit, in which ETB receptor activation induces the release of β-endorphin from keratinocytes and the activation of G-protein-coupled inwardly rectifying potassium channels (GIRKs, also named Kir-3) linked to opioid receptors on nociceptors. These results indicate the existence of an intrinsic feedback mechanism to control peripheral pain in skin, and establish keratinocytes as an ETB receptor–operated opioid pool.

ENDOTHELIN RECEPTORS AND PAIN

UNLABELLED The endogenous endothelin (ET) peptides participate in a remarkable variety of pain-relatedprocesses. Pain that is elevated by inflammation, by skin incision, by cancer, during a Sickle Cell Disease crisis and by treatments that mimic neuropathic and inflammatory pain and are all reduced by local administration of antagonists of endothelin receptors. Many effects of endogenously released endothelin are simulated by acute, local subcutaneous administration of endothelin, which at very high concentrations causes pain and at lower concentrations sensitizes the nocifensive reactions to mechanical, thermal and chemical stimuli. PERSPECTIVE In this paper we review the biochemistry, second messenger pathways and hetero-receptor coupling that are activated by ET receptors, the cellular physiological responses to ET receptor activation, and the contribution to pain of such mechanisms occurring in the periphery and the CNS. Our goal is to frame the subject of endothelin and pain for a broad readership, and to present the generally accepted as well as the disputed concepts, including important unanswered questions.

Tactile allodynia initiated by local subcutaneous endothelin-1 is prolonged by activation of TRPV-1 receptors

Subcutaneous endothelin-1 (ET-1; 200 μM, 2 nmoles/paw) injected into the rat hind paw, has been shown to cause robust hind paw flinching (HPF) and paw licking, and to induce impulses selectively in primary nociceptors. Here we report that a much lower [ET-1] sensitizes the paw to a nocifensive withdrawal response to tactile stimulation (by von Frey hairs, VFH), a sensitization that involves local TRPV1 receptors. Injection of 10 μM ET-1 (0.1 nmole/paw) causes only marginal HPF but rapidly (20 mins after injection) lowers the force threshold for paw withdrawal (PWT) to VFH, to ~30% of pre-injection baseline. Such tactile allodynia persists for 3 hrs. In rats pre-injected with the TRPV1-antagonists capsazepine (CPZ; 1.33 mM) or 5′-iodoresiniferatoxin (I-RTX; 0.13 μM), 15 min before ET-1, a fast initial drop in PWT, as with ET-1 alone, occurs (to 40% or to 19% of baseline, respectively), but this earliest reduction then regresses back to the pre-injection PWT value more rapidly than with ET-1 alone. The recovery of allodynia from the maximum value is about two times faster for ET-1+CPZ and about 4 times faster for ET-1+ I-RTX, compared with that from ET-1 +vehicle (t½ = 130, 60, and 250 mins, respectively). In contrast, spontaneous pain indicated by overt HPF from ET-1 is not attenuated by TRPV1 antagonists. Tactile allodynia is similarly abbreviated by antagonists of both ETA (BQ-123, 32 nmoles/paw) and ETB (BQ-788, 30 nmoles/paw) receptors, whereas HPF is abolished by this ETA antagonist but enhanced by the ETB antagonist. We conclude that low ET-1 causes tactile allodynia, which is characterized by a different time-course and pharmacology than ET-1-induced nociception, and that local TRPV1 receptors are involved in the maintenance of this ET-1-induced allodynia but not in the overt algesic action of ET-1.

The addition of dilute epinephrine produces equieffectiveness of bupivacaine enantiomers for cutaneous analgesia in the rat

We investigated the effectiveness for cutaneous analgesia of bupivacaine (Bup) stereoisomers in male rats. As a model of infiltration anesthesia, inhibition of a nocifensive reflex by subcutaneous injection of 0.6 mL of different concentrations of R-, S-, and racemic-Bup was evaluated quantitatively by the fraction of times a pinprick failed to evoke a nocifensive motor response. R-Bup was more potent in the extent of block; however, S-Bup had a longer-lasting action at smaller doses. This significant difference was apparent when R-Bup and S-Bup were administered in equipotent doses of 0.06% and 0.075%, respectively. Co-injection of epinephrine (Epi) with these equipotent doses enhanced and prolonged the blocking effects of both Bup stereoisomers, although at dilutions of 1:100,000 to 1:1,000,000 Epi itself induced partial, transient analgesia. At 1:2,000,000 dilution, Epi alone had no analgesic effect; however, when co-injected with the shorter-acting R-Bup (0.06%), Epi prolonged its blocking effect to equal the duration of block evoked by equipotent S-Bup (0.075%). We conclude R-Bup is more potent for cutaneous analgesia and that the longer duration of block by S-Bup probably originates from vasoconstrictor activity. Implications Here we show that the more potent optical R-isomer of bupivacaine (Bup) can be used at a smaller dose (80%) than the S-isomer of Bup to give equal pain relief of a skin prick. Although the analgesia from R-Bup is briefer than that from equipotent S-Bup solutions, the durations become equal when a very dilute solution of the vasoconstrictor epinephrine is mixed with the R-isomer. The resulting vasoconstriction thus reduces vascular drug uptake and peak blood levels of systemic drug, reducing potential toxicity.

Sensory fibers resistant to the actions of tetrodotoxin mediate nocifensive responses to local administration of endothelin-1 in rats

&NA; Endothelin‐1 (ET‐1) applied to the sciatic nerve or injected into the plantar hindpaw of rats induces pain behavior (ipsilateral hindpaw flinching) and selective excitation of nociceptors by activation of endothelin‐A (ETA) receptors. To determine the pharmacological profile of the sensory fibers that mediate this pain behavior, we administered lidocaine (LID, a non‐selective conduction blocker) or tetrodotoxin (TTX) prior to ET‐1. LID (1 or 2%, 0.1 ml) was injected percutaneously into the sciatic notch, or TTX (10 &mgr;M, 4 &mgr;l) was injected into the sciatic nerve prior to the more distal application of ET‐1 (400 &mgr;M, 40 &mgr;l) onto the sciatic nerve or subcutaneously into the plantar hindpaw (400 &mgr;M, 10 &mgr;l). LID inhibited ET‐1‐induced flinching in a dose‐dependent manner; the mean total number of flinches was reduced by 39% for 1% LID and by 87% for 2% LID. In contrast, TTX failed to inhibit flinching behavior induced by sciatic nerve application of ET‐1 despite a similar magnitude of motor and sensory blockade as that observed with 2% LID. Partial blockade of flinching behavior by intraneural TTX (mean total flinches were reduced by 51%) was observed after subcutaneous injection of ET‐1. Unexpectedly, ET‐1 prolonged the actions of 1% LID and, even when applied alone, produced clear signs of motor and sensory conduction block. These results are evidence that ET‐1‐induced pain is transmitted to the central nervous system via sensory fibers using tetrodotoxin‐resistant sodium channels, and that ET‐1 has analgesic actions that exist despite the activation of local pain pathways.

NEW PERSPECTIVES ON THE ENDOTHELIN AXIS IN PAIN

The endogenous peptide endothelin (ET) and its several receptors are now well-known participants in a broad variety of conditions that cause pain. Ample evidence supports a direct role for these molecules in causing overt pain and in sensitizing the nervous system to elevated responses to painful stimuli. Our laboratory has recently published an extensive review on the role of endothelins and their receptors (the “endothelin axis”) on pain that occurs under many conditions including inflammation, nerve injury and cancer [1]. In the present review, we highlight some of the key novel findings over the past two years with regard to the biochemistry and molecular pharmacology of endothelin receptors, the diverse intracellular signaling pathways that might couple ET receptor activation to pain-related processes and the newest and updated findings on the in vivo pharmacology of endothelin in various pain states.

Early and Late Contributions of Glutamate and CGRP to Mechanical Sensitization by Endothelin-1

UNLABELLED Intraplantar injection of endothelin-1 (ET-1) (1.5-10 muM) in the rat produces mechanical allodynia. Here we identify the receptor subtypes for ET-1, glutamate and CGRP critical to such allodynia. Antagonism of ET(A) or ET(B) receptors alone, by BQ123 or BQ788, respectively, only partially suppressed allodynia; the combined antagonists prevented allodynia, showing the involvement of both receptor subtypes. Co-injection of NMDA receptor antagonists, (+)MK-801 or D-AP5, with ET-1 also prevented allodynia. In contrast, co-injection of the CGRP1 antagonist CGRP(8-37) attenuated only the later phase of allodynia (>30 min). A mechanistic basis for these effects is shown by ET-1s ability to enhance basal release from cultured sensory neurons of glutamate and CGRP (2.4-fold and 5.7-fold, respectively, for 10 nM ET-1). ET(A) blockade reduced ET-1s enhancement of basal CGRP release by approximately 80%, but basal glutamate release by only approximately 30%. ET-1 also enhanced the capsaicin-stimulated release of CGRP (up to 2-fold for 0.3 nM ET-1), but did not change capsaicin-stimulated glutamate release. Release stimulated by elevated K+ was not altered by ET(A) blockade, nor did blockade of ET(B) reduce any type of release. Thus, ET-1 may induce release of glutamate and CGRP from nerve terminals innervating skin, thereby sensitizing primary afferents, accounting for ET-1-dependent tactile allodynia. PERSPECTIVE The endogenous endothelin peptides participate in a remarkable variety of pain-related processes. The present results provide evidence for the participation of ionotropic glutamatergic receptors and CGRP receptors in the hyperalgesic responses to exogenous ET-1 and suggest clinically relevant targets for further study of elevated pain caused by release of endogenous ET-1.

Sensitization of cutaneous neuronal purinergic receptors contributes to endothelin-1-induced mechanical hypersensitivity.

Summary ETA receptor activation sensitizes neuronal P2X receptors independent of an increase in [Ca2+] and results in mechanical hypersensitivity that involves endogenous ATP release. ABSTRACT Endothelin (ET‐1), an endogenous peptide with a prominent role in cutaneous pain, causes mechanical hypersensitivity in the rat hind paw, partly through mechanisms involving local release of algogenic molecules in the skin. The present study investigated involvement of cutaneous ATP, which contributes to pain in numerous animal models. Pre‐exposure of ND7/104 immortalized sensory neurons to ET‐1 (30 nM) for 10 min increased the proportion of cells responding to ATP (2 &mgr;M) with an increase in intracellular calcium, an effect prevented by the ETA receptor–selective antagonist BQ‐123. ET‐1 (3 nM) pre‐exposure also increased the proportion of isolated mouse dorsal root ganglion neurons responding to ATP (0.2–0.4 &mgr;M). Blocking ET‐1‐evoked increases in intracellular calcium with the IP3 receptor antagonist 2‐APB did not inhibit sensitization to ATP, indicating a mechanism independent of ET‐1‐mediated intracellular calcium increases. ET‐1‐sensitized ATP calcium responses were largely abolished in the absence of extracellular calcium, implicating ionotropic P2X receptors. Experiments using quantitative polymerase chain reaction and receptor‐selective ligands in ND7/104 showed that ET‐1‐induced sensitization most likely involves the P2X4 receptor subtype. ET‐1‐sensitized calcium responses to ATP were strongly inhibited by broad‐spectrum (TNP‐ATP) and P2X4‐selective (5‐BDBD) antagonists, but not antagonists for other P2X subtypes. TNP‐ATP and 5‐BDBD also significantly inhibited ET‐1‐induced mechanical sensitization in the rat hind paw, supporting a role for purinergic receptor sensitization in vivo. These data provide evidence that mechanical hypersensitivity caused by cutaneous ET‐1 involves an increase in the neuronal sensitivity to ATP in the skin, possibly due to sensitization of P2X4 receptors.

Contralateral paw sensitization following injection of endothelin-1: effects of local anesthetics differentiate peripheral and central processes.

Subcutaneous injection of the peptide endothelin-1 (ET-1) into the rats footpad is known to cause rapid, transient ipsilateral mechanical and thermal sensitization and nocifensive hind paw flinching. Here we report that local injection of ET-1 (2 nmoles) into one hind paw slowly sensitizes the contralateral paw to chemical and mechanical stimulation. There was a 1.5-2-fold increase in the hind paw flinching response, over that from the first injection, to a second injection of the same dose of ET-1 delivered 24 h later into the contralateral paw. A similar increase in the number of flinches during the second phase of the response to formalin also occurred in the contralateral paw 24 h after ET-1. The contralateral paw withdrawal threshold to von Frey hairs was lowered by approximately 55% at 24 h after ipsilateral ET-1 injection. ET-1 injected s.c. at a segmentally unrelated location, the nuchal midline, caused no sensitization of the paws, obviating a systemic route of action. Local anesthetic block of the ipsilateral sciatic nerve during the period of initial response to ipsilateral ET-1 prevented contralateral sensitization, indicating the importance of local afferent transmission, although ipsilateral desensitization was not changed. These findings suggest that peripheral ET-1 actions lead to central sensitization that alters responses to selected stimuli.

LOCAL ANTINOCICEPTION INDUCED BY ENDOTHELIN-1 IN THE HAIRY SKIN OF THE RAT’S BACK

UNLABELLED Subcutaneous injection of endothelin-1 (ET-1) into the glabrous skin of the rats hind paw is known to produce impulses in nociceptors and acute nocifensive behavioral responses, such as hind paw flinching, and to sensitize the skin to mechanical and thermal stimulation. In this report, we show that in contrast to the responses in glabrous skin, ET-1 injected subcutaneously into rat hairy skin causes transient antinociception. Concentrations of 1 to 50 microM ET-1 (in 0.05 mL) depress the local nocifensive response to noxious tactile probing at the injection site with von Frey filaments for 30 to 180 minutes; distant injections have no effect at this site, showing that the response is local. Selective inhibition of ET(A) but not of ET(B) receptors inhibits this antinociception, as does coinjection with nimodipine (40 muM), a blocker of L-type Ca(2+) channels. Local subcutaneous injection of epinephrine (45 microM) also causes antinociception through alpha-1 adrenoreceptors, but such receptors are not involved in the ET-1-induced effect. Both epinephrine and ET-1, at antinociceptive concentrations, reduce blood flow in the skin; the effect from ET-1 is largely prevented by subcutaneous nimodipine. These data suggest that ET-1-induced antinociception in the hairy skin of the rat involves cutaneous vasoconstriction, presumably through neural ischemia, resulting in conduction block. PERSPECTIVE The pain-inducing effects of ET-1 have been well documented in glabrous skin of the rat, a frequently used test site. The opposite behavioral effect, antinociception, occurs from ET-1 in hairy skin and is correlated with a reduction in blood flow. Vasoactive effects are important in assessing mechanisms of peripherally acting agents.