Distribution of antinociceptive adenosine A1 receptors in the spinal cord dorsal horn, and relationship to primary afferents and neuronal subpopulations.

Adenosine can reduce pain and allodynia in animals and man, probably via spinal adenosine A1 receptors. In the present study, we investigate the distribution of the adenosine A1 receptor in the rat spinal cord dorsal horn using immunohistochemistry, in situ hybridization, radioligand binding, and confocal microscopy. In the lumbar cord dorsal horn, dense immunoreactivity was seen in the inner part of lamina II. This was unaltered by dorsal root section or thoracic cord hemisection. Confocal microscopy of the dorsal horn revealed close anatomical relationships but no or only minor overlap between A1 receptors and immunoreactivity for markers associated with primary afferent central endings: calcitonin gene-related peptide, or isolectin B4, or with neuronal subpopulations: mu-opioid receptor, neuronal nitric oxide synthase, met-enkephalin, parvalbumin, or protein kinase Cgamma, or with glial cells: glial fibrillary acidic protein. A few adenosine A1 receptor positive structures were double-labeled with alpha-amino-3-hydroxy-5-methyl-4-isoaxolepropionic acid glutamate receptor subunits 1 and 2/3. The results indicate that most of the adenosine A1 receptors in the dorsal horn are located in inner lamina II postsynaptic neuronal cell bodies and processes whose functional and neurochemical identity is so far unknown. Many adenosine A1 receptor positive structures are in close contact with isolectin B4 positive C-fiber primary afferents and/or postsynaptic structures containing components of importance for the modulation of nociceptive information.

Synthesis and opioid activity of [D-Pro10]dynorphin A-(1-11) analogues with N-terminal alkyl substitution.

Several N-terminal di- and monoalkylated derivatives of [D-Pro10]dynorphin A-(1-11) were synthesized in order to explore the structure-activity relationships for antagonist vs agonist activity at kappa-opioid receptors. N,N-Dialkylated and N-monoalkylated (alkyl = allyl, benzyl, and cyclopropylmethyl (CPM) tyrosine derivatives were prepared from tyrosine tert-butyl ester and the corresponding alkyl halides. [D-Pro10]Dyn A-(2-11) was prepared by solid phase synthesis using Fmoc-protected amino acids, and the tyrosine derivatives were coupled to the peptide with BOP ((benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate). Both the degree of substitution and the identity of the alkyl group affected kappa-receptor affinity, selectivity, and efficacy. All of the N-monoalkylated derivatives exhibited much higher affinity (Ki < 0.05 nM) for kappa receptors in the guinea pig cerebellum and greatly enhanced kappa-receptor selectivity (Ki ratio (kappa/mu) > 200) compared to the N,N-dialkyl [D-Pro10]Dyn A-(1-11) analogues, although one disubstituted analogue, N,N-diCPM[D-Pro10]Dyn A-(1-11), retained high affinity (Ki = 0.19 nM) for kappa receptors. Thus the introduction of the second alkyl group at the N-terminus lowered kappa-receptor affinity and selectivity. The N-allyl and N-CPM analogues were moderately potent agonists in the guinea pig ileum (GPI) assay, while the N-benzyl derivative was a weak agonist in this assay. In vivo in the phenylquinone abdominal stretching assay the N-CPM analogue exhibited potent antinociceptive activity (ED50 = 1.1 micrograms/mouse), while N-allyl[D-Pro10]Dyn A-(1-11) exhibited weak antinociceptive activity (ED50 = 27 micrograms/mouse). For the N,N-dialkyl derivatives the identity of the N-terminal alkyl group affected the efficacy observed in the smooth muscle assays. The N,N-diCPM analogue exhibited negligible agonist activity, and N,N-diallyl[D-Pro10]Dyn A-(1-11) showed weak antagonist activity against Dyn A-(1-13)NH2 in the GPI. In contrast, the N,N-dibenzyl compound showed appreciable opioid agonist activity in this assay. In vivo the N,N-diallyl analogue exhibited weak antinociceptive activity (ED50 = 26 micrograms/mouse in the phenylquinone abdominal stretching assay). The N-monoalkylated peptides are among the most kappa-selective opioid peptides reported to date, showing comparable or greater selectivity and higher affinity than the kappa-selective non-peptide agonists U-50,488 and U-69,593. The N,N-diCPM and N,N-diallyl peptides are lead compounds in the development of peptide-based kappa-receptor antagonists.

Synthesis and opioid activity of conformationally constrained dynorphin A analogues. 2. Conformational constraint in the "address" sequence.

Several cyclic lactam analogues of Dyn A-(1-13)NH2 were prepared in order to reduce the conformational flexibility in different regions of the native linear peptide. Cyclo[D-Asp(i),Dap(i+3)]Dyn A-(1-13)NH2 (Dap = alpha,beta-diaminopropionic acid) analogues were designed on the basis of molecular modeling using AMBER, which suggested that this constraint may be compatible with an alpha-helix. The cyclic portion of these constrained analogues spanned from residues 3 to 9, a region proposed by Schwyzer (Biochemistry 1986, 25, 4281) to adopt a helical conformation at kappa receptor sites. Analogues containing Dab (alpha,gamma-diaminobutyric acid) or Orn in position i + 3 were also synthesized to examine the effects of larger ring size. The cyclic peptides exhibited marked differences in binding affinities for kappa, mu, and delta receptors and in opioid activity in the guinea pig ileum (GPI). Cyclo[D-Asp6,Dap9]Dyn A-(1-13)NH2 showed both high kappa receptor affinity and potent agonist activity in the GPI, while cyclo[D-Asp3,Dap6]Dyn A-(1-13)NH2 exhibited very weak binding affinity at all opioid receptors as well as very weak opioid activity in the GPI. Cyclo[D-Asp5,Dap8]Dyn A-(1-13)NH2 showed moderate binding affinity for kappa receptors and was the most kappa selective ligand in this study, but this peptide exhibited very weak agonist activity in the GPI assay. Compared to the corresponding linear peptides, all of the cyclic peptides exhibited decreased mu receptor affinity, while kappa receptor affinity was retained or improved. Therefore the corresponding linear peptides were generally mu selective while the cyclic constrained peptides demonstrated slight selectivity for kappa vs mu receptors or were nonselective. Increasing the ring size by incorporating Dab or Orn in positions 6, 8, or 9 did not significantly affect the binding affinity for the three opioid receptor types nor the opioid activity observed in the GPI. Circular dichroism spectra of the cyclo[D-Asp(i),Dap(i+3)] derivatives in 80% trifluoroethanol at 25 and 5 degrees C suggested differences in the stability of a helical structure when the constraint was incorporated near the N-terminus vs in the middle of the peptide.

Temporal changes in spinal cord expression of mRNA for substance P, dynorphin and enkephalin in a model of chronic pain.

We have used a partial sciatic nerve ligation model to examine the time course for changes in the expression of mRNA for three peptides related to pain transmission at spinal sites (dynorphin, enkephalin and substance P), during the development of allodynia. Enhanced expression of mRNA for dynorphin and substance P was observed in the dorsal horn on the same side as the partial nerve ligation. Increased expression of dynorphin mRNA was biphasic. The initial increases in expression of dynorphin mRNA occurred at 3 h, and a secondary peak was observed 1-3 days after surgery. The secondary increases coincided roughly with increased substance P mRNA expression. However, both dynorphin and substance P mRNA returned to control values after 1 week despite continuing allodynia. No significant changes in expression of mRNA for enkephalin were observed. The elevation of substance P mRNA in intrinsic spinal cord neurons may be secondary to changes in immediate early genes c-fos and jun-B, whereas the expression of dynorphin and enkephalin mRNA is differently regulated. The results also suggest that changes in the expression of the three neuropeptides are not critically involved in the development and maintenance of chronic pain or allodynia.

Spinal expression of mRNA for immediate early genes in a model of chronic pain.

We have used a partial nerve ligation model of chronic pain to investigate if there are changes in the expression of mRNA for several immediate early genes (IEG) that correlate in time with the initial adaptive behavioural changes and with development of allodynia in this model. The animals were inspected for typical changes in posture, and mechanical allodynia was evaluated using von Frey filaments. Expression of three of the immediate early genes examined, c-fos, NGFI-A and jun B, was transiently increased in the ipsilateral dorsal horn of the spinal cord following the partial ligation of the sciatic nerve. The time course and extent of these changes were similar to those reported for acute noxious stimuli. c-jun mRNA expression was significantly enhanced, after a delay of more than 12 h, and then remained elevated over the entire studied period of 4 weeks. These changes occurred only in the ventral horn, particularly in lamina IX. Except for c-jun mRNA, all changes were transient despite behavioural evidence for continuing allodynia. These results from the partial nerve ligation model, when compared with results obtained using other models of acute or chronic nerve injury, suggest that the immediate early genes we have examined are not sufficient to explain the transition to chronic pain states. The results also show that in this model of chronic pain there are prolonged adaptive changes in motor neurons and that these changes are temporally associated with the development of chronic pain and allodynia.

Altered sensory behaviors in mice following manipulation of endogenous spinal adenosine neurotransmission.

Adenosine or adenosine analogs injected intrathecally (i.t.) induce significant antinociception. Recent studies support the existence of an endogenous spinal system that can modulate nociceptive input by releasing adenosine. Inhibition of adenosine metabolism by administration of an adenosine kinase inhibitor, in the present study, decreased behavior induced by putative pain neurotransmitters providing additional support for an endogenous purinergic system. Conversely, administration of high doses of methylxanthines (i.t.), adenosine receptor antagonists, induced behavior similar to that induced by pain neurotransmitters. Methylxanthine (i.t.)-induced behavior was partially inhibited by antagonists of receptors for pain neurotransmitters. These observations are consistent with the hypothesis that an endogenous purinergic system tonically modulates nociceptive input involving a variety of chemical mediators. Preliminary studies also revealed methylxanthine-induced allodynia and suggested spinal purinergic systems may have a broader role in discriminating sensory input.

Time-dependent antinociceptive interactions between opioids and nucleoside transport inhibitors.

Endogenous purinergic systems are important in spinal mechanisms of antinociception. Antinociception induced by spinal mu opioid receptor-selective agonists, in particular, appears to be mediated in part by opioid-stimulated adenosine release. Nucleoside transport system(s) have been implicated both in adenosine release and in its reuptake at spinal sites. The present investigations were designed to determine the significance of nucleoside transport system(s) inhibition in vivo in antinociception induced by opioids administered intrathecally in mice. Dilazep, but not dipyridamole or s (4-nitrobenzyl)-6-thioinosine, nucleoside transport system(s) inhibitors, induced time- and dose-dependent antinociception in the tail-flick test, putatively via spinal adenosine reuptake inhibition. Each nucleoside transport system(s) inhibitor, at doses that have no significant effects alone, enhanced adenosine-mediated antinociception when coadministered intrathecally. Concurrent treatment of mice with opioid receptor-selective agonists and nucleoside transport system(s) inhibitors had varying effects on antinociception, depending on the timing of the nucleoside transport inhibitor. In general, antinociception induced by mu opioid receptor-selective agonists was inhibited by pretreatment, was not affected after coadministration and was enhanced by post-treatment, with nucleoside transport system(s) inhibitors. In contrast, antinociception induced by delta opioid receptor-selective agonists was enhanced by nucleoside transport system(s) inhibitors in all treatment protocols. These results provide in vivo evidence that alterations in adenosine movements into or out of spinal neurons via the nucleoside transport systems can induce antinociception and enhance or inhibit opioid-mediated antinociception. These data also support the hypothesis that adenosine plays significant but independent roles in antinociception induced by mu and delta opioid receptor-selective agonists.

Synthesis and opioid activity of conformationally constrained dynorphin A analogues. 1. Conformational constraint in the "message" sequence.

A constrained analogue of the opioid peptide dynorphin A (Dyn A) cyclized in the "message" sequence was designed which may be compatible with the helical conformation proposed by Schwyzer (Biochemistry 1986, 25, 4281-4286) as the conformation Dyn A adopts at kappa opioid receptors. On the basis of molecular modeling with AMBER, we prepared the lactam cyclo-[D-Asp2,Dap5]Dyn A-(1-13)NH2 (1; Dap = alpha, beta-diaminopropionic acid) containing a four-atom bridge between positions 2 and 5 as a possible constraint compatible with an alpha-helix, along with the homologues with five-(2) and six-atom (3) bridges containing Dab (alpha, gamma-diaminobutyric acid) and Orn, respectively, in position 5. All of the cyclic peptide analogues exhibited high binding affinity for both kappa and mu receptors and high potency in the guinea pig ileum (GPI) assay. As ring size increased, a trend in receptor selectivity from slightly kappa selective (compound 1) to nonselective for kappa vs mu (compound 2) to slightly mu selective (compound 3) was observed in the radioligand binding assays. The results in the GPI for antagonism of these peptides by naloxone paralleled the results of the binding assays and indicated that compound 1 preferentially interacted with kappa receptors in this tissue. Novel byproducts were also obtained from the cyclization reactions with HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and characterized as [D-Asp2,X(Tmg)5]Dyn A-(1-13)NH2 (where X = Dap, Dab, or Orn and Tmg = tetramethylguanidinium). All of the Tmg linear byproducts bound with high affinity to kappa and mu receptors and also exhibited potent agonist activity in the GPI. Circular dichroism spectra of compound 1 and the parent peptide Dyn A-(1-13)NH2 determined in 80% trifluoroethanol at 5 degrees C were consistent with some alpha-helical content in the peptides; comparison of the delta epsilon at 222 nm suggested that compound 1 possessed slightly higher helical content than Dyn A-(1-13)NH2 under these experimental conditions. The cyclic Dyn A analogues 1-3 described here represent the first Dyn A analogues constrained in the "message" sequence with demonstrated high affinity and potency at kappa receptors.

Adenosine kinase and adenosine deaminase inhibition modulate spinal adenosine- and opioid agonist-induced antinociception in mice.

Endogenous purinergic systems mediating antinociception, and their interactions with opioids, were characterized following intrathecal (i.t.) administration of inhibitors of adenosine clearance in mice. 5'-Amino,5'-deoxyadenosine (5'-NH2dAdo), an inhibitor of adenosine kinase, induced significant antinociception after i.t. injection and enhanced antinociception induced by selected opioids (i.t.). Isobolographic analysis of antinociception following coadministration (i.t.) of 5'-NH2dAdo with opioids revealed additive interactions with mu-, and synergistic interactions with delta-, opioid receptor-selective agonists. Inhibitors of adenosine deaminase, deoxycoformycin and erythro-9-(2-hydroxy-3nonyl) adenine (EHNA), generally failed to induce antinociception when administered (i.t.) alone or to enhance opioid (i.t.)-induced antinociception, however, was significantly enhanced by either 5'-NH2dAdo or deoxycoformycin. These results confirm different physiologic roles for adenosine kinase and adenosine deaminase in spinal purinergic systems. 5'-NH2dAdo interactions with opioid receptor-selective agonists demonstrate significant, but heterogeneous interactions between endogenous adenosine and opioid spinal systems mediating antinociception.

Synthesis and opioid activity of dynorphin A-(1-13)NH2 analogues containing cis- and trans-4-aminocyclohexanecarboxylic acid.

It has been proposed that the "message" sequence of dynorphin A (Dyn A) exists in an extended conformation in aqueous solution (Schiller, P. W. Int. J. Pept. Protein Res. 1983, 21, 307-312). Molecular modeling suggested that trans-4-aminocyclohexanecarboxylic acid (trans-ACCA) might function as a conformationally constrained replacement for Gly2-Gly3 of Dyn A in such an extended conformation. ACCA was synthesized by catalytic hydrogenation of p-aminobenzoic acid, and the cis and trans isomers were separated by fractional recrystallization. Analogues of Dyn A-(1-13)-NH2 containing cis- and trans-ACCA were prepared by solid-phase peptide synthesis using the Fmoc chemical protocol. Results from radioligand binding assays indicated that the peptides have modest affinity for kappa opioid receptors (Ki's = 9.1 and 13.4 nM for [cis-ACCA2-3]- and [trans-ACCA2-3]Dyn A-(1-13)NH2, respectively) and modest kappa-receptor selectivity (Ki ratio (kappa/mu/delta) = 1/13/210 and 1/21/103, respectively). [cis-ACCA2-3]- and [trans-ACCA2-3]Dyn A-(1-13)-NH2 are the first reported Dyn A analogues constrained in the "message" sequence that are selective for kappa receptors. The cis-ACCA analogue showed very weak opioid activity (IC50 = 4.0 microM) in the guinea pig ileum.

Involvement of adenosine in antinociception produced by spinal or supraspinal receptor-selective opioid agonists: dissociation from gastrointestinal effects in mice.

Possible involvement of adenosine, as a secondary neurotransmitter, in opioid modulation of nociception and gastrointestinal function was investigated in mice. Inhibitory actions of theophylline, a nonselective adenosine receptor antagonist, were evaluated against effects evoked by opioid receptor-selective agonists administered at spinal or supraspinal sites. Intrathecal administration of theophylline significantly inhibited antinociceptive actions produced by intrathecal (i.th.) injections of morphine, [D-Ala2, NMPhe4, Gly-ol] enkephalin (DAMGO), [D-Pen2, D-Pen5] enkephalin (DPDPE) and beta-endorphin as measured with the warm water tail-flick assay. The rank order of rightward displacement of i.th. agonist dose-response curves by theophylline (i.th.) was DPDPE (greatest) > DAMGO > morphine > beta-endorphin. Theophylline was less effective as an inhibitor in the hot-plate assay. Additionally, i.th. administration of theophylline inhibited antinociceptive effects evoked by i.c.v. administration of opioids. The rank order of rightward displacement of dose-response curves after i.c.v. opioid administration was DAMGO (greatest) > beta-endorphin > morphine > DPDPE. In contrast to the effectiveness of theophylline administered i.th., theophylline coadministered i.c.v. with opioid agonists did not inhibit opioid-induced antinociception. Neither i.th. nor i.c.v. theophylline altered inhibitory effects on gastric emptying and gastrointestinal propulsion produced by i.th. or i.c.v. administration of selective opioid agonists. These data provide additional support for involvement of spinal adenosine as a secondary neurotransmitter in opioid antinociceptive processes associated with local spinal reflexes as well as in descending antinociceptive processes. Adenosine was not involved in modulation of opioid-activated gastrointestinal outflow pathways at either spinal or supraspinal levels.

Effect of modification of the basic residues of dynorphin A-(1-13) amide on kappa opioid receptor selectivity and opioid activity.

A series of dynorphin A-(1-13) amide (Dyn A-(1-13)NH2) analogues containing lysine or N epsilon-acetyllysine (Lys(Ac)) was prepared by solid-phase peptide synthesis and evaluated for opioid receptor affinity in radioligand binding assays and for opioid activity in the guinea pig ileum (GPI). Substitutions were made at positions 6, 7, 9, 11, and 13, the basic amino acids in the C-terminus of the peptide, in order to assess the individual contributions of these residues to the kappa opioid receptor affinity and selectivity of Dyn A-(1-13)NH2. While substitutions of Lys(Ac) for Arg in position 6 did not affect kappa receptor affinity, it enhanced affinity for mu and delta receptors and therefore caused a loss of kappa receptor selectivity. When Lys(Ac) was substituted for Arg9, kappa opioid receptor affinity was enhanced and kappa receptor selectivity was retained. Replacement for Arg7, Lys11, or Lys13 by Lys(Ac) resulted in both decreased affinity and selectivity for kappa receptors. These results demonstrate the importance of Arg6 to the receptor selectivity profile of Dyn A-(1-13)NH2 and indicate that, of the five basic residues in the C-terminus, only Arg9 can be replaced by a nonbasic residue without substantial loss of kappa opioid receptor selectivity.

Synthesis and opioid activity of 2-substituted dynorphin A-(1-13) amide analogues.

A series of 2-substituted dynorphin A-(1-13) amide (Dyn A-(1-13)NH2) analogues was prepared by solid phase peptide synthesis and evaluated for opioid receptor affinities in radioligand binding assays and for opioid activity in the guinea pig ileum (GPI) assay. Amino acid substitution at the 2 position produced marked differences in both opioid receptor affinities and potency in the GPI assay; Ki values for the analogues in the radioligand binding assays and IC50 values in the GPI assay varied over three to four orders of magnitude. The parent peptide, Dyn A-(1-13)NH2, exhibited the greatest affinity and selectivity for kappa receptors and was the most potent peptide examined in the GPI assay. The most important determinant of opioid receptor selectivity and opioid potency for the synthetic analogues was the stereochemistry of the amino acid at the 2 position. Except for [D-Lys2]Dyn A-(1-13)NH2 in the kappa receptor binding assay, the analogues containing a D-amino acid at position 2 were much more potent in all of the assays than their corresponding isomers containing an L-amino acid at this position. The L-amino acid-substituted analogues generally retained some selectivity for kappa opioid receptors. The more potent derivatives with a D-amino acid in position 2, however, preferentially interacted with mu opioid receptors. Introduction of a positively charged amino acid into the 2 position generally decreased opioid receptor affinities and potency in the GPI assay.

Spinally-mediated antinociception is induced in mice by an adenosine kinase-, but not by an adenosine deaminase-, inhibitor.

Relative involvement of adenosine deaminase and adenosine kinase in antinociception induced by endogenous adenosine was investigated. Antinociception induced by 5'-amino 5'-deoxyadenosine (5'-ADAdo; an adenosine kinase inhibitor) and deoxycoformycin (dCF; an adenosine deaminase inhibitor) administered i.t. was determined using the mouse tail-flick assay. Dose- and time-dependent antinociception was observed following i.t. administration of 5'-ADAdo, but not dCF. Antinociception induced by 5'-ADAdo was reversed by coadministration i.t. of theophylline, an adenosine receptor antagonist, in a dose-dependent manner. These data provide preliminary evidence that adenosine kinase plays a more significant physiological role than adenosine deaminase in the regulation of adenosine involved in spinally-mediated antinociception.

Descending systems activated by morphine (ICV) inhibit kainic acid (IT)-induced behavior.

Modulation of spinal systems activated by N-methyl-D-aspartate (NMDA) and substance P administered IT have been an area of interest in several laboratories. In the present investigations, behavior induced by the excitatory amino acid kainic acid, but not quisqualate, is demonstrated to be modulated in a manner similar to that previously observed for NMDA. Biting, scratching and licking behavior was induced by IT injections of excitatory amino acids or substance P in mice. Behavior induced by kainic acid (IT) injection was inhibited in a dose-dependent manner by coadministration of morphine (ICV), norepinephrine (IT), N-ethyl carboxamidoadenosine (NECA) (IT) and agonists interacting at PCP receptors (IT). Kainic acid and NMDA differed, however, in that a dopaminergic agonist, apomorphine, inhibited kainic acid-, but not NMDA-induced behavior and a selective NMDA receptor antagonist inhibits NMDA-, but not kainic acid-induced behavior. Behavior induced by quisqualate (IT) was not inhibited by any treatment and may have nonspecific actions in this type of assay. Our observations support independent spinal sites of action for behavior induced by kainic acid and NMDA, but several similarities were observed in the modulation of spinal systems activated by these agents.

Morphine (intracerebroventricular) activates spinal systems to inhibit behavior induced by putative pain neurotransmitters.

Previous investigations find that morphine administered i.c.v. induces antinociception directly at supraspinal sites and indirectly via activation of descending spinal systems. Independent experimentation suggests substance P and N-methyl-D-aspartate (NMDA) administered intrathecally (i.t.) can act as putative pain neurotransmitters to stimulate afferent pathways mediating nociception. The present studies were designed to determine whether a functional link exists between these observations. Mice were administered morphine i.c.v. 15 min before i.t. injections of substance P or NMDA. Additional investigations utilized coadministration of substance P or NMDA i.t. with one of several antagonists. Morphine administered i.c.v. inhibited both substance P- and NMDA-induced behavior in a dose-dependent manner. Coadministration of noradrenergic or adenosine receptor antagonists with substance P or NMDA i.t. dose-dependently reversed morphine (i.c.v.)-mediated inhibition. Methysergide injected i.t. caused significant, but only partially effective, antagonism of the effects of morphine (i.c.v.). Naloxone coadministered i.t. was effective in reversing morphine (i.c.v.)-mediated inhibition of NMDA-induced behavior, but ineffective in the substance P assay. These data demonstrate a functional link between activation of descending systems mediating antinociception by morphine (i.c.v.) and inhibition of putative pain neurotransmitters by spinally active antinociceptive agents. The potential involvement of serotonergic and opioid spinal systems is not clear, but noradrenergic and adenosine spinal pathways appear to play an important role in the indirect actions of morphine (i.c.v.). Differences in the inhibition of NMDA- and substance P-induced behavior also provide evidence for the presence of substance P and NMDA receptors in separate afferent pathways transmitting nociceptive stimuli.

In vivo characterization of phencyclidine/sigma agonist-mediated inhibition of nociception.

Substance P and excitatory amino acids have been implicated as potential nociceptive neurotransmitters in several investigations. Excitatory amino acids acting at N-methyl-D-aspartate (NMDA) receptors are of particular interest because of the description of NMDA/phencyclidine (PCP) receptor complexes. PCP receptors are one of two populations of receptors resolved from a population previously referred to as 'sigma opioid' receptors. Agonists, including sigma opioid agonists, interacting with PCP receptors non-competitively inhibit NMDA-induced effects. Therefore, it has been suggested that NMDA/PCP receptor complexes in nociceptive systems may explain the antinociceptive effects of sigma opioid agonists. In the present studies, highly selective ligands for PCP and sigma receptors were coadministered with NMDA or substance P i.t. The rank order potency for inhibition of NMDA-induced behavior was (+/-)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate (MK-801) greater than PCP greater than (+/-)N-allyl-normetazocine ((+/-)-SKF10,047). 1,3-Di-ortho-tolyl-guanidine (DTG) and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+/-)-3PPP) were inactive. Inhibition of NMDA-induced behavior by PCP receptor agonists was not reversed by haloperidol, a putative sigma receptor antagonist. These data support PCP, but not sigma, receptor-mediated inhibition of behavior induced by NMDA. Behavior induced by i.t. administration of substance P was similarly inhibited by PCP receptor agonists, but inhibition could be reversed by coadministration of haloperidol or (+)-butaclamol. These data suggest a dopaminergic mechanism for PCP inhibition of substance P-induced behavior. Our results confirm the existence of NMDA/PCP receptor complexes in spinal systems mediating nociception and suggest agonists may induce antinociception by interacting with spinal PCP receptors.(ABSTRACT TRUNCATED AT 250 WORDS)