GABAA receptor-mediated excitation of nociceptive afferents in the rat isolated spinal cord-tail preparation.

Algogens such as capsaicin, bradykinin, acetylcholine, 5-hydroxytryptamine and potassium ions applied to exposed tail skin of the rat isolated spinal cord-tail preparation evoke a ventral root response consisting of depolarization and spiking activity. L-glutamate and kainate also evoke similar reflexes. All these compounds evoke depolarization of afferent axons or dorsal root ganglion cells. Since GABA depolarizes unmyelinated afferent fibers, the ability of GABA receptor agonists to activate cutaneous nociceptive afferents has been examined. GABA superfused over exposed tail skin evoked a ventral root reflex essentially identical to that produced by capsaicin (3 microM). The EC50 was 27 microM. Muscimol, 3-aminopropane sulphonate, isoguvacine and beta-alanine had effects comparable to GABA, with EC50 values of 9.6, 26, 56 and 870 microM respectively. Baclofen (100 microM) or glycine (10 mM) had no effect. Bicuculline applied to the tail competitively antagonized GABA (Schild slope = -1.03) with a pA2 of 5.8. Spinal application of 1 microM morphine blocked the actions of GABA and capsaicin. These data indicate that GABAA receptors can depolarize and excite nociceptive afferents. GABA could be involved in nociception by contributing to firing of C-fibres, or by analogy to presynaptic inhibition in the spinal cord, may act to decrease neuropeptide transmitter release in cutaneous tissue.

WIN 63480, a hydrophilic TCP-site ligand, has reduced agonist-independent NMDA ion channel access compared to MK-801 and phencyclidine.

NMDA channel blockers are potentially advantageous therapeutic agents for the treatment of ischemia and head trauma, which greatly elevate extracellular glutamate, because they should most effectively inhibit high levels of receptor activation. A novel high affinity TCP site ligand, WIN 63480, does not produce MK-801- or PCP-like behavioral activation at anti-ischemic doses. While WIN 63480, MK-801 and PCP were all observed to be effective blockers of open NMDA channels, WIN 63480 had much less access to closed NMDA channels. This difference may be due to the fact that WIN 63480 is hydrophilic (logD = -4.1) while MK-801 and PCP are lipophilic (logD = +1.8). In vivo, closed channel access may result in a non-competitive profile of antagonism for MK-801 and PCP compared to a more uncompetitive profile for WIN 63480. Release of glutamate, and depolarization, are likely to produce a high level of NMDA receptor activation in ischemic areas compared to normal tissue. Consequently, at anti-ischemic doses, WIN 63480 may produce less inhibition of physiological NMDA-mediated processes in neural systems involved in behavioral regulation than MK-801 or PCP, leading to an improved side effect profile.

Lack of correlation between sigma binding potency and inhibition of contractions in the mouse vas deferens preparation.

The existence of sigma receptors in the mouse, rat and guinea pig vasa deferentia has previously been proposed, although drug effects are inconsistent and generally occur only at high concentrations. The purpose of the present study was to evaluate lower, physiologically relevant concentrations of ligands for possible sigma effects on electrically stimulated twitch contractions in the mouse vas deferens (MVD). Putative sigma agonists and antagonists all inhibited 0.1 Hz electrically stimulated twitch contractions in nM concentrations. Inhibitory activity plateaued between 20 and 60% for all compounds except 1,3-di(2-tolyl)guanidine (DTG), which had a shallow concentration-effect curve. Subsequent to the plateau, higher concentrations (30 microM) of rimcazole and haloperidol fully inhibited electrically stimulated twitch contractions. There was no correlation between inhibitory potency or maximal effect in the MVD and binding potency at sigma sites in either MVD or guinea pig brain. The inhibitory effects of R(+)-3-(3-hydroxyphenyl)-N-1-propylpiperidine ((+)3-PPP) or DTG on electrically stimulated twitch contractions were not antagonized by the putative sigma antagonists DTG, haloperidol, rimcazole or BMY-14802, nor by alpha 2-adrenoceptor, dopamine D1, dopamine D2 or opiate antagonists. Although the mechanism of sigma ligand effects in the MVD has not been established, the data caution against a presumption that effects of sigma ligands on electrically stimulated twitch contractions in this preparation are mediated by sigma receptors.

Activation of nociceptive reflexes by peripheral kainate receptors.

The rat isolated spinal cord-tail preparation was used to examine the ability of peripherally applied excitatory amino acids to stimulate ventral root reflexes. Bolus applications (3 sec) of domoate (0.1-10 microM) or kainate (KA; 10-300 microM) to exposed skin of the rat tail stimulated ventral root responses comparably to those evoked by capsaicin (0.3-10 microM) or bradykinin (0.1-10 microM). The ventral root potential evoked by both capsaicin and KA is thought to be a nociceptive response because both compounds selectively activate peripheral C-fibers. L-Glutamate (0.01-10 mM), quisqualate (0.1-1 mM) and amino-5-methyl-4-isoxazole-propionic acid (0.1-1.0 mM) were of lower potency than KA, whereas N-methyl-D-aspartate (1 mM) and L-aspartate (10 mM) were inactive. Responses evoked by prolonged application of KA faded rapidly. Capsaicin-evoked responses partially faded during prolonged application, but residual spiking activity was recorded for at least 30 min. KA did not evoke any observable response during application of capsaicin. The effects of KA and capsaicin were blocked by spinal application of morphine (0.3-1.0 microM) in a naloxone-reversible manner, consistent with activation of peripheral nociceptive afferents. The action of KA, but not capsaicin, was competitively inhibited by the amino-5-methyl-4-isoxazole-propionic acid/KA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (10-100 microM) applied to the rat tail (pA2 = 5.9). Conversely, ruthenium red (1 microM) selectively blocked capsaicin-evoked nociceptive reflexes. The existence of functional KA receptors on nociceptive afferents suggests that peripherally released glutamate or other excitatory amino acids could be involved in nociception or neurogenic inflammation.

L-glutamate activates peripheral nociceptors.

The chemosensitivity of nociceptive afferents has been investigated using the isolated spinal-cord-tail preparation of the neonatal rat. L-glutamate applied to exposed rat tail skin stimulated nociceptive reflexes (ED50 = 136 microM). The action of L-glutamate was selective, since other constitutive L-amino acids or D-glutamate were inactive at a concentration of 10 mM. The known algogens bradykinin, acetylcholine, serotonin and potassium ions also stimulated nociceptive reflexes. Histamine (1 mM) and substance P (0.1 mM) had no observable effect. L-glutamate, which is released from macrophages, could therefore contribute to inflammation-related nociception by stimulation of cutaneous nociceptive nerve endings.