Actions of capsaicin on peripheral nociceptors of the neonatal rat spinal cord-tail in vitro: dependence of extracellular ions and independence of second messengers.

1. We have tested the hypothesis that capsaicin-induced activation, desensitization and impairment of peripheral nociceptor function is mediated by separate mechanisms. This was investigated by use of an in vitro preparation of the neonatal rat spinal cord with the functionally attached tail in which the cord and tail were separately superfused with physiological solution. Activation of peripheral fibres by noxious (capsaicin, bradykinin, 5-hydroxytrptamine, heat, pinch) and innocuous (light brush) stimuli was assessed by recording the depolarization of a spinal ventral root (L3-L5). 2. Brief administration of capsaicin produced dose-related depolarizing responses (EC50 = 280 nM). These responses could be reproduced for many hours following the repeated application of capsaicin at a submaximal concentration. Prolonged application of 0.5-2.0 microM capsaicin induced a selective desensitization to subsequent brief administrations of capsaicin. Prolonged administration at 20-50 microM produced an additional non-selective reduction in responses to all noxious stimuli without changing innocuous brush responses. 3. Removal of extracellular calcium from the tail superfusate did not reduce the response to capsaicin or prevent capsaicin-induced desensitization. However, high concentrations of capsaicin no longer induced a non-specific reduction of responses to other noxious stimuli. The response to a brief administration of capsaicin was unaffected by calcium channel blocking drugs including nifedipine, cadmium or omega-conotoxin. On the other hand high extracellular calcium increased the incidence of the non-selective reduction of responses to all noxious stimuli produced by high concentrations of capsaicin. 4. Replacement of extracellular sodium with choline blocked peripheral nerve conduction but did not prevent the desensitization produced by capsaicin. In addition, high concentrations of capsaicin were less effective in reducing the responsiveness to other noxious stimuli. 5. Neither capsaicin-evoked responses nor capsaicin-induced desensitization were affected by the administration of forskolin, dibutyryl cyclic AMP, nitroprusside, dibutyryl cyclic GMP, beta-12,13 phorbol dibutyrate, trifluoperazine, indomethacin, staurosporine or mepacrine, in the tail superfusate. 6. These data suggest that capsaicin-induced activation, desensitization and impairment of peripheral nociceptors may be separable phenomena. Extracellular calcium is not required for capsaicin-induced activation or desensitization but calcium as well as sodium are important for capsaicin-induced impairment of nociceptive responses. Desensitization may occur independently of peripheral fibre activation and cannot be attributed to a central mechanism. Finally neither capsaicin-induced activation nor desensitization require the participation of a second messenger.

NE-19550 and NE-21610, antinociceptive capsaicin analogues: studies on nociceptive fibres of the neonatal rat tail in vitro.

When applied to peripheral fibres in a neonatal rat tail/spinal cord preparation in vitro, capsaicin (0.2-50 microM) induced an activation, selective desensitization and reduced responses to other noxious stimuli (heat, bradykinin). Similar concentrations of the antinociceptive analogues NE-19550 and NE-21610, did not affect peripheral fibre responsiveness but induced cross desensitization to capsaicin. At 500 microM both analogues produced similar effects to capsaicin. Capsaicin analogues may induce analgesia without initial activation of nociceptors.

Opioid receptor ligands in the neonatal rat spinal cord: binding and in vitro depression of the nociceptive responses.

1. Opioid receptors in the neonatal rat spinal cord have been characterized by measurements of ligand binding to crude membrane fractions and by functional tests on the nociceptive spinal response in a spinal cord-tail preparation in vitro. 2. There were high affinity binding sites for [3H]-[D-Ala2, MePhe4, Gly(ol)5]enkephalin (DAGOL), [3H]-U69593, and [3H]-ethylketocyclazocine (EKC) on spinal cord membranes from neonatal rats. Hill slopes for binding of [3H]-DAGOL and [3H]-U69593 were close to unity. The Hill slope for binding of [3H]-EKC was less than unity, even after its interactions at mu-receptors had been blocked with 100 nM unlabelled DAGOL. Binding sites for [3H]-[D-Pen2, D-Pen5]enkephalin (DPDPE) could not be detected. 3. In competition assays U50488 was as potent as PD117302 and U69593 in competition for either [3H]-U69593 or [3H]-EKC binding sites. Hill slopes for a range of competing ligands at [3H]-DAGOL or [3H]-U69593 sites were close to unity. Hill slopes for competition at [3H]-EKC sites were less than one. 4. In the spinal cord-tail preparation from neonatal rats, opioid receptor agonists depressed spinal nociceptive responses evoked by application of capsaicin or heat to the tail. The order of potency was DAGOL greater than U69593 = PD117302 greater than morphine greater than U50488 = [D-Pen2, L-Pen5]enkephalin (DPLPE). 5. The antagonist naloxone was about equally potent against DAGOL, morphine and DPLPE, and about ten times less potent against U69593 and PD117302. The effects of U50488 were much less sensitive to blockade by naloxone than the effects of PD11703 or U69593. The Kappa antagonist, nor-binaltorphimine was equipotent against all three Kappa agonists. 6. The absence of delta-binding sites, and the low potency and relatively high sensitivity to naloxone suggest that DPLPE could be working at mu-receptors in the neonatal rat spinal cord. 7. The binding assays show that U50488 has the same affinity as PD1 17302 and U69593 for Kappa-receptors, yet it was less effective in the depression of nociceptive responses. This may be because U50488 has a relatively low efficacy at Kappa-receptors. It is possible that at high concentrations U50488 activates receptors not affected by other Kappa-ligands. These additional receptors may be non-opioid receptors (hence the insensitivity to naloxone), or they could be a subtype of Kappa-opioid receptor.

Resiniferatoxin, a potent capsaicin-like stimulator of peripheral nociceptors in the neonatal rat tail in vitro.

1. A spinal ventral root response was measured following the activation of peripheral fibres by noxious (heat at 48 degrees C, capsaicin, bradykinin) and innocuous (brush) stimuli in a preparation of the neonatal rat spinal cord-tail maintained in vitro. 2. Following superfusion of the tail with 0.1-1.0 nM of the potent irritant, resiniferatoxin (RTX), brief, irregular depolarization and a selective loss of capsaicin sensitivity was produced. RTX 10-100 nM evoked a tonic response, initiated transient irregular depolarizations and densitization to further applications of RTX and capsaicin but not to other stimuli. Following RTX 1 microM a prolonged loss of sensitivity to all noxious stimuli was produced. 3. When a selective densitization to capsaicin was produced by a long application of capsaicin, RTX was also ineffective. 4. Superfusion of the tail with 4 beta-phorbol, 12, 13-dibutyrate (PDBu), a protein kinase C activator, stimulated capsaicin-sensitive peripheral fibres. Prolonged administration of PDBu attenuated or abolished further responses to PDBu and bradykinin but responses to RTX and capsaicin were unchanged. The protein kinase C inhibitor staurosporine (50-200 nM), attenuated the effects of PDBu and bradykinin but not those of RTX or capsaicin. 5. The present data suggest that neither RTX nor capsaicin act on peripheral nociceptors via a phorbol ester-like stimulation of protein kinase C. Rather, RTX acts on nociceptors by a similar mechanism to capsaicin. These effects may be the basis for the irritant properties of RTX and may further relate to the antinociceptive actions observed in vivo. RTX is therefore a potent new tool with which to investigate the properties of nociceptive neurones and provides a prototype for further development of antinociceptive agents.

Capsaicin desensitization of peripheral nociceptive fibres does not impair sensitivity to other noxious stimuli.

Capsaicin was tested on peripheral fibres in vitro to determine whether evoked depolarization or desensitization were likely to explain the antinociception observed after acute systemic capsaicin in vivo. The activation of peripheral fibres by noxious (capsaicin, bradykinin, heat) and innocuous (light brush) stimuli was recorded as a depolarization of a spinal ventral root (L3-L5) in the neonatal rat spinal cord with attached tail. Prolonged superfusion of the tail with low doses (0.2-2 microM) of capsaicin produced a short lasting depolarization followed by a complete loss of sensitivity to capsaicin without changes in sensitivity to other noxious or innocuous stimuli. Partial recovery from this selective desensitization could be observed 3-5 h later. In most preparations superfusion of the tail with 20 microM capsaicin produced a prolonged and non-selective impairment of sensitivity to all noxious stimuli. These data suggest that neither depolarization by capsaicin nor the selective desensitization of peripheral fibres to capsaicin are likely to account for the acute antinociceptive effect of systemic capsaicin. On the other hand the non-selective reduction in sensitivity to noxious stimuli induced by capsaicin may contribute to its antinociceptive action.

Activation of a bradykinin receptor in peripheral nerve and spinal cord in the neonatal rat in vitro.

In an in vitro preparation of the neonatal rat spinal cord with attached tail, administration of bradykinin (Bk) to the spinal cord or to the tail produced depolarization of a ventral root (L3-L5). The effect of Bk at each site was selectively and reversibly antagonized by D-Arg [Hyp2, Thi5,8 D-Phe7]-Bk but could not be mimicked or antagonized by the B1-receptor ligands [des-Arg9]-Bk or Leu8[des-Arg9]-Bk, respectively. Peripherally evoked noxious responses produced by capsaicin or heat, were unaffected by either antagonist administered to the spinal cord. These data suggest that Bk-evoked responses in the spinal cord and at peripheral nociceptors were mediated via a receptor which by definition is of the B2-type. Additionally Bk is unlikely to be a physiological mediator of acute nociception in the spinal cord.

Bradykinin-induced stimulation of afferent fibres is mediated through protein kinase C.

In an in vitro preparation of the neonatal spinal cord with the tail attached, brief administration of bradykinin or capsaicin in the tail superfusate containing a normal calcium concentration, activated peripheral fibres and produced a depolarization recorded at a spinal ventral root (L3-L5). Perfusion with a phorbol ester (4 beta-phorbol 12,13-dibutyrate, PDBu) produced a small and inconsistent activation of peripheral fibres. In subsequent experiments calcium was omitted from the tail superfusate since under this condition responses to bradykinin and capsaicin were unchanged but PDBu evoked reproducible depolarization when applied at intervals of 60 min or more. Prolonged desensitization followed repeated administration at shorter intervals. Pretreatment of the tail with capsaicin, to impair transmission in C-fibres, abolished the effect of each agonist. Inactivation of protein kinase C with the inhibitor staurosporine (10-100 nM) attenuated the effect of bradykinin and PDBu but not that of capsaicin. Pretreatment with PDBu also attenuated the effect of bradykinin. These data suggest that a phorbol ester and bradykinin stimulate capsaicin-sensitive C-fibres by a mechanism which involves the activation of protein kinase C.